Dispersion and detachment of cell aggregates

ABSTRACT

Compositions comprising a protein or isolated peptide, and methods using the same for preventing, dispersing or detaching a biofilm, are disclosed.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 14/123,455(National Stage of PCT/IB2012/001061) filed Apr. 4, 2014, which claimspriority from U.S. Provisional Application 61/491,756, filed May 31,2011. The aforesaid applications are incorporated herein by reference intheir entirety.

This application contains a Sequence Listing which has been submitted inASCII format via EFS-WEB and is hereby incorporated by reference in itsentirety. Said ASCII copy created on Nov. 27, 2013, is namedsequence.txt and is 60 KB.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for dispersingor detaching a single cell organism from a surface or from other cellsor single cell organisms, especially where the organism is in a biofilmor in vivo.

BACKGROUND OF THE INVENTION

Microorganisms can live and proliferate as individual cells swimmingfreely in the environment (e.g., plankton), or they can grow as highlyorganized, multicellular communities encased in a self-producedpolymeric matrix in close association with surfaces and interfaces. Thelatter microbial lifestyle is referred to as biofilms. Biofilm formationrepresents an ancient, protected mode of growth that allows microbialsurvival in hostile environments and allows microorganisms to disperseand colonize new niches [Hall-Stoodley et al., Nat Rev Microbiol. (2004)2(2):95-108]. The composition of biofilms is complex and variable amongdifferent microbial species and even within the same species underdifferent environmental conditions. Nonetheless, biofilm formationrepresents the normal lifestyle of microorganism in the environment andall microbes can make biofilms. Previous studies revealed that bacterialbiofilm formation progresses through multiple developmental stagesdiffering in protein profiles [Sauer et al, J Bacteriol. (2002) 184(4):1140-54], beginning with attachment to surface, followed by theimmigration and division to form microcolonies and finally maturationinvolving expression of matrix polymers. Bacteria within each biofilmstage display phenotypes and possess properties that are markedlydifferent from those of the same group growing planktonically [Sauer etal., J Bacteriol. (2004) 186(21):7312-26]. Biofilms are a major cause ofsystemic infections (e.g., nosocomial infections) in humans.

The composition of biofilms is complex and variable among differentmicrobial species and even within the same species under differentenvironmental conditions. Nonetheless, biofilm formation represents thenormal lifestyle of microorganism in the environment and all microbescan make biofilms. Previous studies revealed that bacterial biofilmformation progresses through multiple developmental stages differing inprotein profiles [Sauer et al., J Bacteriol. (2002) 184(4): 1140-54],beginning with attachment to surface, followed by the immigration anddivision to form microcolonies and finally maturation involvingexpression of matrix polymers. Bacteria within each biofilm stagedisplay phenotypes and possess properties that are markedly differentfrom those of the same group growing planktonically [Sauer et al., JBacteriol. (2004) 186(21):7312-26].

In the body, biofilms can be associated with tissues (e.g., inner ears,teeth, gums, lungs, heart valves and the urogenital tract) and can be amajor source of systemic infections. An estimated 65% of bacterialinfections in humans are biofilm in nature. Additionally, after formingbiofilms, microorganisms tend to change their characteristics, sometimesdrastically, such that doses of antibiotics which normally kill theorganisms in suspended cultures are completely ineffective against thesame microorganisms when the organisms are in attached or conglomeratebiofilm form. See U.S. Pat. No. 7,189,351, incorporated by reference inits entirety.

One of the principal concerns with respect to products that areintroduced into the body (e.g., contact lenses, central venouscatheters, mechanical heart valves and pacemakers) or provide a pathwayinto the body is microbial infection and invariably biofilm formation.As these infections are difficult to treat with antibiotics, removal ofthe device is often necessitated, which is traumatic to the patient andincreases the medical cost. PCT Application No. WO 06/006172 disclosesthe use of anti-amyloid agents, such as aromatic compounds, forinhibiting formation or disintegrating a pre existing biofilm. Theapplication discloses that compounds preventing amyloid fibril formationin Alzheimers can act against fibril formation in biofilms, andconcludes that amino acids having an aromatic arm are effective againstbiofilms. However, the analysis was limited to full length sequences.

Biofilms can cause, amongst a wide range of negative effects,accelerated corrosion in industrial systems, oil souring and biofouling.Bacterial aggregation can occur in agriculture [Monier et al., Appliedand Environmental Microbiology, 70(1): 346-355 (2004); Biofilms in thefood and beverage industries, Edited by P M Fratamico, B A Annous and NW Guenther, USDA ARS, USA, Woodhead Publishing Series in Food Science,Technology and Nutrition No. 181, Chapter 20, pages 517-535] and inwater systems [Carlson et al., Zentralbl Bakteriol Orig B, 161(3):233-247 (1975)]. Biofouling may be caused by the adhesion of organismsto any surface in a marine or freshwater environment, including coolingtowers, water pipes and filters in cooling or desalinizationinstallations, irrigation and power stations, and membranes, such asthose used in wastewater and desalinization systems. Biofouling alsooccurs in aquaculture systems in fish farms. Furthermore the commercialshipping fleets of the world consume approximately 300 million tons offuel annually. Without antifouling measures, that fuel consumption wouldincrease by as much as 40%, equivalent to an extra 120 million tonnes offuel annually. The economic cost of this was estimated as about $7.5billion in 2000; a more recent estimate is $30 billion. Generally,biofilms are very difficult to eliminate since microbes growing withinare highly organized and can withstand hostile environments, such ashigh temperatures and anti-microbial agents (e.g., antibiotics).

Since marine-aquatic plants and animals are continuously exposed to alarge diversity and abundance of potentially harmful microorganisms inthe form of biofilm, and it is known that marine life produceanti-microbial peptides, it is possible that broad spectrum naturalfactors that interfere with biofilm formation may also be present inmarine life.

U.S. Publication No. 20070098745 discloses means of preventing biofilmformation by the use of reef fish microflora. This invention describesanti-biofilm substances derived from bacteria isolated from theepithelial mucosal surfaces of healthy coral reef fish (e.g., Sparisomaninidae and Lutjanus purpureus). The bacterial isolates produce signalsor toxins that prevent biofilm formation.

Cell clustering is not limited to microbial biofilms, but can exist invivo. Alzheimer's Disease, for example, involves neuron clusters (i.e.,neuritic plaques) in the brain [Tiraboschi et al., J. Neurology, 62(11):1984-1989 (2004)]. In the body, bacterial aggregation can occur orally[Duffau et al., 16 Sep. 2005 RAI Congress, #0299; Liljemark et al.,Infect. Immun., 31(3): 935-941 (1981)]; in sepsis [Reid et al., CurrentMicrobiology, 20(3): 185-190 (1990)]; diarrhea [Bieber et al., Science,280(5372): 2114-2118 (1998)]; in nosocomial infections [Bortz et al.,Bulletin of Mathematical Biology, Volume 70, Number 3, 745-768]; inrelation to drug efficacy [Kraal et al., J Dent Res 58(11): 2125-2131(1979)]; in relation to peritoneal dialysis [Reid et al., PeritonealDialysis International, 10: 21-24 (1990)]; lung diseases [Sanchez etal., PLoS Pathog 6(8)]; and Crohn's disease [Isenmann et al., DigestiveDiseases and Sciences, 47(2): 462-468 (2002)].

Cell clustering can also occur among white blood cells in vivo. Forexample, white blood cells can aggregate in whole blood as the result ofcigarette smoking and lead to microvascular occlusion and damage [Hillet al., J. R. Soc. Med., 86(3):139-140 (1993)]. White blood cellaggregation can also occur in vascular disease [Belch et al., ThrombosisResearch, 48(6):631-639 (1987)]. Macrophage-lymphocyte clustering iscorrelated to rheumatoid arthritis [Webb et al., Macrophage-lymphocyteclustering in rheumatoid arthritis, Ann. rheum. Dis. (1975), 34, 38]Additionally, Sun et al. state, “Both platelet aggregation and whiteblood cell aggregation are involved in pathological processes such asthrombosis, atherosclerosis and chronic inflammation. People in olderage groups are likely to suffer from cardiovascular diseases and mayhave increased white cell and platelet aggregation which couldcontribute to this increased risk.” [Sun et al., A study of whole bloodplatelet and white cell aggregation using a laser flow aggregometer,Platelets (2003) March 14(2):103-8.] Furthermore, adhesion andaggregation of white blood cells are involved in vascular diseases andthrombosis [Belch et al., Whole blood white cell aggregation: a noveltechnique, Thrombosis Research, 48; 631-639 (1987)].

Cell clustering also occurs in restenosis, which can develop as theresult of implanted medical stents [Dangas et al., Circulation, 105:2586(2005)]. Such clustering can lead to the occlusion of a blood vessel anddramatically reduced blood flow. One of the symptoms of the second stageof restenosis, which tends to occur 3-6 months after surgery, isplatelet aggregation at the site of the injury [Michael Kirchengast*,Klaus Munter. Endothelin and restenosis. Cardiovascular Research 39(1998) 550-555] and residual plaque burden outside the stent [Prati etal., In-Stent Neointimal Proliferation Correlates With the Amount ofResidual Plaque Burden Outside the Stent. An Intravascular UltrasoundStudy, Circulation, (1999) 99:1011-1014.], both phenomena being the maincauses of in-stent neointimal proliferation. Patri et al concludes withthe following: “Late in-stent neointimal proliferation has a directcorrelation with the amount of residual plaque burden after coronarystent implantation, supporting the hypothesis that plaque removal beforestent implantation may reduce restenosis.”

SUMMARY OF THE INVENTION

The present invention provides a peptide consisting of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 1), wherein X¹ is S,N, I, V, R, K, Q or L; X² is V, I, A, N, L or Q; X³ is P; X⁴ is Y, F orW; X⁵ is D, N, Q or E; X⁶ is Y, F, R, W, V, H, L, K or I; X⁷ is N, S, G,D, H, E, Q or I; X⁸ is W, L, F, M S, T, R, A, G, V, P, Y, I or K; X⁹ isY, N, F, K, L, R, I, V, W or Q; X¹⁰ is S, K, N, T, E, R, L, Q, I, V, D,or K; X¹¹ is N, E, D, Q, S, A or I; and X¹² is W, R, V, L, I, K, F or E,wherein the peptide is not SVPYDYNWYSNW (SEQ ID NO: 2). The presentinvention also provides a peptide consisting of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 3), wherein X¹ is S,N, T, K, R, H, E, I, Q or D; X² is V, I, L, Y, G, F or W; X³ is H, N, Q,E, D or S; X⁴ is S, P, A or T; X⁵ is F, W or Y; X⁶ is D, N, E or Q; X⁷is Y, F or W; X⁸ is D, G or E; X⁹ is W, F or Y; X¹⁰ is Y, F or W; X¹¹ isN or Q; and X¹² is V, I or L, wherein the peptide is not SVHSFDYDWYNV(SEQ ID NO: 4).

In some embodiments, the peptide is SVPFDYNLYSNW (SEQ ID NO: 5);SAPYNFNFYSNW (SEQ ID NO: 6); NIPFNFSLNKER (SEQ ID NO: 7); SVPYQYNWYSNW(SEQ ID NO: 8); SVPWEYNFYSNW (SEQ ID NO: 9); RIPYDRGMIVNV (SEQ ID NO:10); KVPYDWDSVINL (SEQ ID NO: 11); QLPYDVHTYNDW (SEQ ID NO: 12);LAPYDHNRYTQW (SEQ ID NO: 13); SNPYDLEAYENW (SEQ ID NO: 14); SVPYDYQGYRNI(SEQ ID NO: 15); SVPYDYNVYLNK (SEQ ID NO: 16); IQPYDKNYFQNF (SEQ ID NO:17); VVPYDINIKDNW (SEQ ID NO: 18); SVPYDYNPYSNW (SEQ ID NO: 19);SVPYDYNKLKNW (SEQ ID NO: 20); SVPYDYNWRSSW (SEQ ID NO: 21); SVPYDYNWWSAW(SEQ ID NO: 22); SVPYDYNWQSNW (SEQ ID NO: 23); ELSSFNFDWYNV (SEQ ID NO:24); RYSSFDYDWYNV (SEQ ID NO: 25); NVHSFDYDWYNV (SEQ ID NO: 26);RVESFNYDWYNV (SEQ ID NO: 27); RVESFDFDWYNI (SEQ ID NO: 28); RINSFDYDWYNV(SEQ ID NO: 29); TVNSFDYDWYNV (SEQ ID NO: 30); KVNSFDYDWYNV (SEQ ID NO:31); TVHSFDYDWYNV (SEQ ID NO: 32); SVHSWDYDWYNV (SEQ ID NO: 33);SVHSYDFDWYNV (SEQ ID NO: 34); TLQAFNYEWYQL (SEQ ID NO: 35); KYETFEYGWYNI(SEQ ID NO: 36); HGDSFQYEWYNL (SEQ ID NO: 37); SVHSFDWDWYNV (SEQ ID NO:38); SVHSFDYDYYNV (SEQ ID NO: 39); SVHSFDYDFYNV (SEQ ID NO: 40);SVHSFDYDWFNV (SEQ ID NO: 41); SVHSFDYDWWNV (SEQ ID NO: 42); IFNPFDYDWYNV(SEQ ID NO: 43); QWHSFDYDWYNV (SEQ ID NO: 44) or DVHPFDYDWYNV (SEQ IDNO: 45).

In some embodiments, the peptide is cyclic peptide. In otherembodiments, the peptide is soluble. In some embodiments, the peptide isattached to a linker. In some embodiments, the linker is polyethyleneglycol or palmitic acid. In other embodiments, the peptide is synthetic.

The present invention also provides a composition comprising a proteinor peptide, wherein said composition is capable of detaching a singlecell organism from a surface or from other single cell organisms. Insome embodiments, the peptide consists of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 1), wherein X¹ is S,N, I, V, R, K, Q or L; X² is V, I, A, N, L or Q; X³ is P; X⁴ is Y, F orW; X⁵ is D, N, Q or E; X⁶ is Y, F, R, W, V, H, L, K or I; X⁷ is N, S, G,D, H, E, Q or I; X⁸ is W, L, F, M S, T, R, A, G, V, P, Y, I or K; X⁹ isY, N, F, K, L, R, I, V, W or Q; X¹⁰ is S, K, N, T, E, R, L, Q, I, V, D,or K; X¹¹ is N, E, D, Q, S, A or I; and X¹² is W, R, V, L, I, K, F or E,wherein the peptide is not SVPYDYNWYSNW (SEQ ID NO: 2). In otherembodiments, the peptide consists of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 3), wherein X¹ is S,N, T, K, R, H, E, I, Q or D; X² is V, I, L, Y, G, F or W; X³ is H, N, Q,E, D or S; X⁴ is S, P, A or T; X⁵ is F, W or Y; X⁶ is D, N, E or Q; X⁷is Y, F or W; X⁸ is D, G or E; X⁹ is W, F or Y; X¹⁰ is Y, F or W; X¹¹ isN or Q; and X¹² is V, I or L, wherein the peptide is not SVHSFDYDWYNV(SEQ ID NO: 4).

In specific embodiments, the peptide is SVPFDYNLYSNW (SEQ ID NO: 5);SAPYNFNFYSNW (SEQ ID NO: 6); NIPFNFSLNKER (SEQ ID NO: 7); SVPYQYNWYSNW(SEQ ID NO: 8); SVPWEYNFYSNW (SEQ ID NO: 9); RIPYDRGMIVNV (SEQ ID NO:10); KVPYDWDSVINL (SEQ ID NO: 11); QLPYDVHTYNDW (SEQ ID NO: 12);LAPYDHNRYTQW (SEQ ID NO: 13); SNPYDLEAYENW (SEQ ID NO: 14); SVPYDYQGYRNI(SEQ ID NO: 15); SVPYDYNVYLNK (SEQ ID NO: 16); IQPYDKNYFQNF (SEQ ID NO:17); VVPYDINIKDNW (SEQ ID NO: 18); SVPYDYNPYSNW (SEQ ID NO: 19);SVPYDYNKLKNW (SEQ ID NO: 20); SVPYDYNWRSSW (SEQ ID NO: 21); SVPYDYNWWSAW(SEQ ID NO: 22); SVPYDYNWQSNW (SEQ ID NO: 23); ELSSFNFDWYNV (SEQ ID NO:24); RYSSFDYDWYNV (SEQ ID NO: 25); NVHSFDYDWYNV (SEQ ID NO: 26);RVESFNYDWYNV (SEQ ID NO: 27); RVESFDFDWYNI (SEQ ID NO: 28); RINSFDYDWYNV(SEQ ID NO: 29); TVNSFDYDWYNV (SEQ ID NO: 30); KVNSFDYDWYNV (SEQ ID NO:31); TVHSFDYDWYNV (SEQ ID NO: 32); SVHSWDYDWYNV (SEQ ID NO: 33);SVHSYDFDWYNV (SEQ ID NO: 34); TLQAFNYEWYQL (SEQ ID NO: 35); KYETFEYGWYNI(SEQ ID NO: 36); HGDSFQYEWYNL (SEQ ID NO: 37); SVHSFDWDWYNV (SEQ ID NO:38); SVHSFDYDYYNV (SEQ ID NO: 39); SVHSFDYDFYNV (SEQ ID NO: 40);SVHSFDYDWFNV (SEQ ID NO: 41); SVHSFDYDWWNV (SEQ ID NO: 42); IFNPFDYDWYNV(SEQ ID NO: 43); QWHSFDYDWYNV (SEQ ID NO: 44) or DVHPFDYDWYNV (SEQ IDNO: 45).

In some embodiments, peptide is a cyclic peptide. In some embodiments,the peptide is soluble. In other embodiments, the peptide is attached toa linker. In some embodiments, the linker is polyethylene glycol orpalmitic acid. In some embodiments, the peptide is synthetic. In someembodiments, the organism is in a biofilm. In some embodiments, theorganism is an aquatic microorganism. In some embodiments, the organismsare attached in a cluster or aggregate. In some embodiments, thecomposition is capable of breaking or dispersing said cluster oraggregate. In some embodiments, the detaching affects the ability ofsaid organism to produce polysaccharide matrix. In some embodiments, thesurface is a selected from the group comprising a fabric, a fiber, afoam, a film, a concrete, a masonry, a glass, a metal and a plastic.

The present invention also provides a method of detaching a single cellorganism from a surface or from other single cell organisms, comprisingcontacting said organism with a composition comprising a protein orpeptide. In some embodiments, the peptide consists of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 1), wherein X¹ is S,N, I, V, R, K, Q or L; X² is V, I, A, N, L or Q; X³ is P; X⁴ is Y, F orW; X⁵ is D, N, Q or E; X⁶ is Y, F, R, W, V, H, L, K or I; X⁷ is N, S, G,D, H, E, Q or I; X⁸ is W, L, F, M S, T, R, A, G, V, P, Y, I or K; X⁹ isY, N, F, K, L, R, I, V, W or Q; X¹⁰ is S, K, N, T, E, R, L, Q, I, V, D,or K; X¹¹ is N, E, D, Q, S, A or I; and X¹² is W, R, V, L, I, K, F or E,wherein the peptide is not SVPYDYNWYSNW (SEQ ID NO: 2). In otherembodiments, the peptide consists of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 3), wherein X¹ is S,N, T, K, R, H, E, I, Q or D; X² is V, I, L, Y, G, F or W; X³ is H, N, Q,E, D or S; X⁴ is S, P, A or T; X⁵ is F, W or Y; X⁶ is D, N, E or Q; X⁷is Y, F or W; X⁸ is D, G or E; X⁹ is W, F or Y; X¹⁰ is Y, F or W; X¹¹ isN or Q; and X¹² is V, I or L, wherein the peptide is not SVHSFDYDWYNV(SEQ ID NO: 4).

In specific embodiments, the peptide is SVPFDYNLYSNW (SEQ ID NO: 5);SAPYNFNFYSNW (SEQ ID NO: 6); NIPFNFSLNKER (SEQ ID NO: 7); SVPYQYNWYSNW(SEQ ID NO: 8); SVPWEYNFYSNW (SEQ ID NO: 9); RIPYDRGMIVNV (SEQ ID NO:10); KVPYDWDSVINL (SEQ ID NO: 11); QLPYDVHTYNDW (SEQ ID NO: 12);LAPYDHNRYTQW (SEQ ID NO: 13); SNPYDLEAYENW (SEQ ID NO: 14); SVPYDYQGYRNI(SEQ ID NO: 15); SVPYDYNVYLNK (SEQ ID NO: 16); IQPYDKNYFQNF (SEQ ID NO:17); VVPYDINIKDNW (SEQ ID NO: 18); SVPYDYNPYSNW (SEQ ID NO: 19);SVPYDYNKLKNW (SEQ ID NO: 20); SVPYDYNWRSSW (SEQ ID NO: 21); SVPYDYNWWSAW(SEQ ID NO: 22); SVPYDYNWQSNW (SEQ ID NO: 23); ELSSFNFDWYNV (SEQ ID NO:24); RYSSFDYDWYNV (SEQ ID NO: 25); NVHSFDYDWYNV (SEQ ID NO: 26);RVESFNYDWYNV (SEQ ID NO: 27); RVESFDFDWYNI (SEQ ID NO: 28); RINSFDYDWYNV(SEQ ID NO: 29); TVNSFDYDWYNV (SEQ ID NO: 30); KVNSFDYDWYNV (SEQ ID NO:31); TVHSFDYDWYNV (SEQ ID NO: 32); SVHSWDYDWYNV (SEQ ID NO: 33);SVHSYDFDWYNV (SEQ ID NO: 34); TLQAFNYEWYQL (SEQ ID NO: 35); KYETFEYGWYNI(SEQ ID NO: 36); HGDSFQYEWYNL (SEQ ID NO: 37); SVHSFDWDWYNV (SEQ ID NO:38); SVHSFDYDYYNV (SEQ ID NO: 39); SVHSFDYDFYNV (SEQ ID NO: 40);SVHSFDYDWFNV (SEQ ID NO: 41); SVHSFDYDWWNV (SEQ ID NO: 42); IFNPFDYDWYNV(SEQ ID NO: 43); QWHSFDYDWYNV (SEQ ID NO: 44) or DVHPFDYDWYNV (SEQ IDNO: 45).

In some embodiments, the peptide is a cyclic peptide. In someembodiments, the peptide is soluble. In other embodiments, the peptideis attached to a linker. In some embodiments, the linker is polyethyleneglycol or palmitic acid. In some embodiments, the peptide is synthetic.In some embodiments, the surface is selected from the group comprising afabric, a fiber, a foam, a film, a concrete, a masonry, a glass, a metaland a plastic. In some embodiments, the organism is a in a biofilm. Insome embodiments, the organism is an aquatic microorganism. In someembodiments, the organisms are attached in a cluster or aggregate. Insome embodiments, composition breaks or disperses said cluster oraggregate. In some embodiments, the composition prevents said organismfrom producing polysaccharide matrix. In some embodiments, the surfaceis a selected from the group comprising a fabric, a fiber, a foam, afilm, a concrete, a masonry, a glass, a metal and a plastic.

The present invention also provides a pharmaceutical compositioncomprising a protein or peptide, wherein the composition is capable ofdetaching a single cell organism from a surface or from other singlecell organisms, and a pharmaceutically acceptable carrier or diluent. Insome embodiments, the pharmaceutical composition comprises a peptideconsisting of amino acids X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ IDNO: 1), wherein X¹ is S, N, I, V, R, K, Q or L; X² is V, I, A, N, L orQ; X³ is P; X⁴ is Y, F or W; X⁵ is D, N, Q or E; X⁶ is Y, F, R, W, V, H,L, K or I; X⁷ is N, S, G, D, H, E, Q or I; X⁸ is W, L, F, M S, T, R, A,G, V, P, Y, I or K; X⁹ is Y, N, F, K, L, R, I, V, W or Q; X¹⁰ is S, K,N, T, E, R, L, Q, I, V, D, or K; X¹¹ is N, E, D, Q, S, A or I; and X¹²is W, R, V, L, I, K, F or E, wherein the peptide is not SVPYDYNWYSNW(SEQ ID NO: 2). In other embodiments, the peptide consists of aminoacids X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 3), wherein X¹is S, N, T, K, R, H, E, I, Q or D; X² is V, I, L, Y, G, F or W; X³ is H,N, Q, E, D or S; X⁴ is S, P, A or T; X⁵ is F, W or Y; X⁶ is D, N, E orQ; X⁷ is Y, F or W; X⁸ is D, G or E; X⁹ is W, F or Y; X¹⁰ is Y, F or W;X¹¹ is N or Q; and X¹² is V, I or L, wherein the peptide is notSVHSFDYDWYNV (SEQ ID NO: 4).

In specific embodiments, the peptide is SVPFDYNLYSNW (SEQ ID NO: 5);SAPYNFNFYSNW (SEQ ID NO: 6); NIPFNFSLNKER (SEQ ID NO: 7); SVPYQYNWYSNW(SEQ ID NO: 8); SVPWEYNFYSNW (SEQ ID NO: 9); RIPYDRGMIVNV (SEQ ID NO:10); KVPYDWDSVINL (SEQ ID NO: 11); QLPYDVHTYNDW (SEQ ID NO: 12);LAPYDHNRYTQW (SEQ ID NO: 13); SNPYDLEAYENW (SEQ ID NO: 14); SVPYDYQGYRNI(SEQ ID NO: 15); SVPYDYNVYLNK (SEQ ID NO: 16); IQPYDKNYFQNF (SEQ ID NO:17); VVPYDINIKDNW (SEQ ID NO: 18); SVPYDYNPYSNW (SEQ ID NO: 19);SVPYDYNKLKNW (SEQ ID NO: 20); SVPYDYNWRSSW (SEQ ID NO: 21); SVPYDYNWWSAW(SEQ ID NO: 22); SVPYDYNWQSNW (SEQ ID NO: 23); ELSSFNFDWYNV (SEQ ID NO:24); RYSSFDYDWYNV (SEQ ID NO: 25); NVHSFDYDWYNV (SEQ ID NO: 26);RVESFNYDWYNV (SEQ ID NO: 27); RVESFDFDWYNI (SEQ ID NO: 28); RINSFDYDWYNV(SEQ ID NO: 29); TVNSFDYDWYNV (SEQ ID NO: 30); KVNSFDYDWYNV (SEQ ID NO:31); TVHSFDYDWYNV (SEQ ID NO: 32); SVHSWDYDWYNV (SEQ ID NO: 33);SVHSYDFDWYNV (SEQ ID NO: 34); TLQAFNYEWYQL (SEQ ID NO: 35); KYETFEYGWYNI(SEQ ID NO: 36); HGDSFQYEWYNL (SEQ ID NO: 37); SVHSFDWDWYNV (SEQ ID NO:38); SVHSFDYDYYNV (SEQ ID NO: 39); SVHSFDYDFYNV (SEQ ID NO: 40);SVHSFDYDWFNV (SEQ ID NO: 41); SVHSFDYDWWNV (SEQ ID NO: 42); IFNPFDYDWYNV(SEQ ID NO: 43); QWHSFDYDWYNV (SEQ ID NO: 44) or DVHPFDYDWYNV (SEQ IDNO: 45).

The present invention also provides a method of preventing or treating apathogen infection in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising a protein or peptide, wherein saidcomposition is capable of detaching a single cell organism from asurface or from other single cell organisms. In some embodiments, thepharmaceutical composition comprises a peptide consisting of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 1), wherein X¹ is S,N, I, V, R, K, Q or L; X² is V, I, A, N, L or Q; X³ is P; X⁴ is Y, F orW; X⁵ is D, N, Q or E; X⁶ is Y, F, R, W, V, H, L, K or I; X⁷ is N, S, G,D, H, E, Q or I; X⁸ is W, L, F, M S, T, R, A, G, V, P, Y, I or K; X⁹ isY, N, F, K, L, R, I, V, W or Q; X¹⁰ is S, K, N, T, E, R, L, Q, I, V, D,or K; X¹¹ is N, E, D, Q, S, A or I; and X¹² is W, R, V, L, I, K, F or E,wherein the peptide is not SVPYDYNWYSNW (SEQ ID NO: 2). In otherembodiments, the peptide consists of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 3), wherein X¹ is S,N, T, K, R, H, E, I, Q or D; X² is V, I, L, Y, G, F or W; X³ is H, N, Q,E, D or S; X⁴ is S, P, A or T; X⁵ is F, W or Y; X⁶ is D, N, E or Q; X⁷is Y, F or W; X⁸ is D, G or E; X⁹ is W, F or Y; X¹⁰ is Y, F or W; X¹¹ isN or Q; and X¹² is V, I or L, wherein the peptide is not SVHSFDYDWYNV(SEQ ID NO: 4).

In specific embodiments, the peptide is SVPFDYNLYSNW (SEQ ID NO: 5);SAPYNFNFYSNW (SEQ ID NO: 6); NIPFNFSLNKER (SEQ ID NO: 7); SVPYQYNWYSNW(SEQ ID NO: 8); SVPWEYNFYSNW (SEQ ID NO: 9); RIPYDRGMIVNV (SEQ ID NO:10); KVPYDWDSVINL (SEQ ID NO: 11); QLPYDVHTYNDW (SEQ ID NO: 12);LAPYDHNRYTQW (SEQ ID NO: 13); SNPYDLEAYENW (SEQ ID NO: 14); SVPYDYQGYRNI(SEQ ID NO: 15); SVPYDYNVYLNK (SEQ ID NO: 16); IQPYDKNYFQNF (SEQ ID NO:17); VVPYDINIKDNW (SEQ ID NO: 18); SVPYDYNPYSNW (SEQ ID NO: 19);SVPYDYNKLKNW (SEQ ID NO: 20); SVPYDYNWRSSW (SEQ ID NO: 21); SVPYDYNWWSAW(SEQ ID NO: 22); SVPYDYNWQSNW (SEQ ID NO: 23); ELSSFNFDWYNV (SEQ ID NO:24); RYSSFDYDWYNV (SEQ ID NO: 25); NVHSFDYDWYNV (SEQ ID NO: 26);RVESFNYDWYNV (SEQ ID NO: 27); RVESFDFDWYNI (SEQ ID NO: 28); RINSFDYDWYNV(SEQ ID NO: 29); TVNSFDYDWYNV (SEQ ID NO: 30); KVNSFDYDWYNV (SEQ ID NO:31); TVHSFDYDWYNV (SEQ ID NO: 32); SVHSWDYDWYNV (SEQ ID NO: 33);SVHSYDFDWYNV (SEQ ID NO: 34); TLQAFNYEWYQL (SEQ ID NO: 35); KYETFEYGWYNI(SEQ ID NO: 36); HGDSFQYEWYNL (SEQ ID NO: 37); SVHSFDWDWYNV (SEQ ID NO:38); SVHSFDYDYYNV (SEQ ID NO: 39); SVHSFDYDFYNV (SEQ ID NO: 40);SVHSFDYDWFNV (SEQ ID NO: 41); SVHSFDYDWWNV (SEQ ID NO: 42); IFNPFDYDWYNV(SEQ ID NO: 43); QWHSFDYDWYNV (SEQ ID NO: 44) or DVHPFDYDWYNV (SEQ IDNO: 45).

The present invention also provides a method of increasing theeffectiveness of a pharmaceutical composition, the method comprisingadministering a composition comprising a protein or peptide, wherein thecomposition is capable of detaching a microorganism from a surface orfrom other microorganisms to a subject in need of the pharmaceuticalcomposition. In some embodiments, the pharmaceutical compositioncomprises a peptide consisting of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 1), wherein X¹ is S,N, I, V, R, K, Q or L; X² is V, I, A, N, L or Q; X³ is P; X⁴ is Y, F orW; X⁵ is D, N, Q or E; X⁶ is Y, F, R, W, V, H, L, K or I; X⁷ is N, S, G,D, H, E, Q or I; X⁸ is W, L, F, M S, T, R, A, G, V, P, Y, I or K; X⁹ isY, N, F, K, L, R, I, V, W or Q; X¹⁰ is S, K, N, T, E, R, L, Q, I, V, D,or K; X¹¹ is N, E, D, Q, S, A or I; and X¹² is W, R, V, L, I, K, F or E,wherein the peptide is not SVPYDYNWYSNW (SEQ ID NO: 2). In otherembodiments, the peptide consists of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 3), wherein X¹ is S,N, T, K, R, H, E, I, Q or D; X² is V, I, L, Y, G, F or W; X³ is H, N, Q,E, D or S; X⁴ is S, P, A or T; X⁵ is F, W or Y; X⁶ is D, N, E or Q; X⁷is Y, F or W; X⁸ is D, G or E; X⁹ is W, F or Y; X¹⁰ is Y, F or W; X¹¹ isN or Q; and X¹² is V, I or L, wherein the peptide is not SVHSFDYDWYNV(SEQ ID NO: 4).

In specific embodiments, the peptide is SVPFDYNLYSNW (SEQ ID NO: 5);SAPYNFNFYSNW (SEQ ID NO: 6); NIPFNFSLNKER (SEQ ID NO: 7); SVPYQYNWYSNW(SEQ ID NO: 8); SVPWEYNFYSNW (SEQ ID NO: 9); RIPYDRGMIVNV (SEQ ID NO:10); KVPYDWDSVINL (SEQ ID NO: 11); QLPYDVHTYNDW (SEQ ID NO: 12);LAPYDHNRYTQW (SEQ ID NO: 13); SNPYDLEAYENW (SEQ ID NO: 14); SVPYDYQGYRNI(SEQ ID NO: 15); SVPYDYNVYLNK (SEQ ID NO: 16); IQPYDKNYFQNF (SEQ ID NO:17); VVPYDINIKDNW (SEQ ID NO: 18); SVPYDYNPYSNW (SEQ ID NO: 19);SVPYDYNKLKNW (SEQ ID NO: 20); SVPYDYNWRSSW (SEQ ID NO: 21); SVPYDYNWWSAW(SEQ ID NO: 22); SVPYDYNWQSNW (SEQ ID NO: 23); ELSSFNFDWYNV (SEQ ID NO:24); RYSSFDYDWYNV (SEQ ID NO: 25); NVHSFDYDWYNV (SEQ ID NO: 26);RVESFNYDWYNV (SEQ ID NO: 27); RVESFDFDWYNI (SEQ ID NO: 28); RINSFDYDWYNV(SEQ ID NO: 29); TVNSFDYDWYNV (SEQ ID NO: 30); KVNSFDYDWYNV (SEQ ID NO:31); TVHSFDYDWYNV (SEQ ID NO: 32); SVHSWDYDWYNV (SEQ ID NO: 33);SVHSYDFDWYNV (SEQ ID NO: 34); TLQAFNYEWYQL (SEQ ID NO: 35); KYETFEYGWYNI(SEQ ID NO: 36); HGDSFQYEWYNL (SEQ ID NO: 37); SVHSFDWDWYNV (SEQ ID NO:38); SVHSFDYDYYNV (SEQ ID NO: 39); SVHSFDYDFYNV (SEQ ID NO: 40);SVHSFDYDWFNV (SEQ ID NO: 41); SVHSFDYDWWNV (SEQ ID NO: 42); IFNPFDYDWYNV(SEQ ID NO: 43); QWHSFDYDWYNV (SEQ ID NO: 44) or DVHPFDYDWYNV (SEQ IDNO: 45).

In some embodiments, the pharmaceutical composition is an antibiotic.

The present invention also provides a method of identifying ananti-biofilm composition, the method comprising: (a) contacting saidbiofilm with a plurality of compositions, each composition comprising aprotein or peptide; (b) assaying the ability of said biofilm to resistanti-biofilm activity, wherein said anti-biofilm activity comprisesdetaching said biofilm from a surface or breaking up said biofilm; and(c) identifying from said plurality of compositions at least onecomposition having said anti-biofilm activity above a predeterminedthreshold, thereby identifying the anti-biofilm composition. In someembodiments, the composition comprises a peptide consisting of aminoacids X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 1), wherein X¹is S, N, I, V, R, K, Q or L; X² is V, I, A, N, L or Q; X³ is P; X⁴ is Y,F or W; X⁵ is D, N, Q or E; X⁶ is Y, F, R, W, V, H, L, K or I; X⁷ is N,S, G, D, H, E, Q or I; X⁸ is W, L, F, M S, T, R, A, G, V, P, Y, I or K;X⁹ is Y, N, F, K, L, R, I, V, W or Q; X¹⁰ is S, K, N, T, E, R, L, Q, I,V, D, or K; X¹¹ is N, E, D, Q, S, A or I; and X¹² is W, R, V, L, I, K, For E, wherein the peptide is not SVPYDYNWYSNW (SEQ ID NO: 2). In otherembodiments, the peptide consists of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 3), wherein X¹ is S,N, T, K, R, H, E, I, Q or D; X² is V, I, L, Y, G, F or W; X³ is H, N, Q,E, D or S; X⁴ is S, P, A or T; X⁵ is F, W or Y; X⁶ is D, N, E or Q; X⁷is Y, F or W; X⁸ is D, G or E; X⁹ is W, F or Y; X¹⁰ is Y, F or W; X¹¹ isN or Q; and X¹² is V, I or L, wherein the peptide is not SVHSFDYDWYNV(SEQ ID NO: 4).

In specific embodiments, the peptide is SVPFDYNLYSNW (SEQ ID NO: 5);SAPYNFNFYSNW (SEQ ID NO: 6); NIPFNFSLNKER (SEQ ID NO: 7); SVPYQYNWYSNW(SEQ ID NO: 8); SVPWEYNFYSNW (SEQ ID NO: 9); RIPYDRGMIVNV (SEQ ID NO:10); KVPYDWDSVINL (SEQ ID NO: 11); QLPYDVHTYNDW (SEQ ID NO: 12);LAPYDHNRYTQW (SEQ ID NO: 13); SNPYDLEAYENW (SEQ ID NO: 14); SVPYDYQGYRNI(SEQ ID NO: 15); SVPYDYNVYLNK (SEQ ID NO: 16); IQPYDKNYFQNF (SEQ ID NO:17); VVPYDINIKDNW (SEQ ID NO: 18); SVPYDYNPYSNW (SEQ ID NO: 19);SVPYDYNKLKNW (SEQ ID NO: 20); SVPYDYNWRSSW (SEQ ID NO: 21); SVPYDYNWWSAW(SEQ ID NO: 22); SVPYDYNWQSNW (SEQ ID NO: 23); ELSSFNFDWYNV (SEQ ID NO:24); RYSSFDYDWYNV (SEQ ID NO: 25); NVHSFDYDWYNV (SEQ ID NO: 26);RVESFNYDWYNV (SEQ ID NO: 27); RVESFDFDWYNI (SEQ ID NO: 28); RINSFDYDWYNV(SEQ ID NO: 29); TVNSFDYDWYNV (SEQ ID NO: 30); KVNSFDYDWYNV (SEQ ID NO:31); TVHSFDYDWYNV (SEQ ID NO: 32); SVHSWDYDWYNV (SEQ ID NO: 33);SVHSYDFDWYNV (SEQ ID NO: 34); TLQAFNYEWYQL (SEQ ID NO: 35); KYETFEYGWYNI(SEQ ID NO: 36); HGDSFQYEWYNL (SEQ ID NO: 37); SVHSFDWDWYNV (SEQ ID NO:38); SVHSFDYDYYNV (SEQ ID NO: 39); SVHSFDYDFYNV (SEQ ID NO: 40);SVHSFDYDWFNV (SEQ ID NO: 41); SVHSFDYDWWNV (SEQ ID NO: 42); IFNPFDYDWYNV(SEQ ID NO: 43); QWHSFDYDWYNV (SEQ ID NO: 44) or DVHPFDYDWYNV (SEQ IDNO: 45).

The present invention also provides a medical device comprising acomposition comprising a protein or peptide, wherein the composition iscapable of detaching a single cell organism from a surface or from othersingle cell organisms. In some embodiments, the medical device comprisesa peptide consisting of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 1), wherein X¹ is S,N, I, V, R, K, Q or L; X² is V, I, A, N, L or Q; X³ is P; X⁴ is Y, F orW; X⁵ is D, N, Q or E; X⁶ is Y, F, R, W, V, H, L, K or I; X⁷ is N, S, G,D, H, E, Q or I; X⁸ is W, L, F, M S, T, R, A, G, V, P, Y, I or K; X⁹ isY, N, F, K, L, R, I, V, W or Q; X¹⁰ is S, K, N, T, E, R, L, Q, I, V, D,or K; X¹¹ is N, E, D, Q, S, A or I; and X¹² is W, R, V, L, I, K, F or E,wherein the peptide is not SVPYDYNWYSNW (SEQ ID NO: 2). In otherembodiments, the peptide consists of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 3), wherein X¹ is S,N, T, K, R, H, E, I, Q or D; X² is V, I, L, Y, G, F or W; X³ is H, N, Q,E, D or S; X⁴ is S, P, A or T; X⁵ is F, W or Y; X⁶ is D, N, E or Q; X⁷is Y, F or W; X⁸ is D, G or E; X⁹ is W, F or Y; X¹⁰ is Y, F or W; X¹¹ isN or Q; and X¹² is V, I or L, wherein the peptide is not SVHSFDYDWYNV(SEQ ID NO: 4).

In specific embodiments, the peptide is SVPFDYNLYSNW (SEQ ID NO: 5);SAPYNFNFYSNW (SEQ ID NO: 6); NIPFNFSLNKER (SEQ ID NO: 7); SVPYQYNWYSNW(SEQ ID NO: 8); SVPWEYNFYSNW (SEQ ID NO: 9); RIPYDRGMIVNV (SEQ ID NO:10); KVPYDWDSVINL (SEQ ID NO: 11); QLPYDVHTYNDW (SEQ ID NO: 12);LAPYDHNRYTQW (SEQ ID NO: 13); SNPYDLEAYENW (SEQ ID NO: 14); SVPYDYQGYRNI(SEQ ID NO: 15); SVPYDYNVYLNK (SEQ ID NO: 16); IQPYDKNYFQNF (SEQ ID NO:17); VVPYDINIKDNW (SEQ ID NO: 18); SVPYDYNPYSNW (SEQ ID NO: 19);SVPYDYNKLKNW (SEQ ID NO: 20); SVPYDYNWRSSW (SEQ ID NO: 21); SVPYDYNWWSAW(SEQ ID NO: 22); SVPYDYNWQSNW (SEQ ID NO: 23); ELSSFNFDWYNV (SEQ ID NO:24); RYSSFDYDWYNV (SEQ ID NO: 25); NVHSFDYDWYNV (SEQ ID NO: 26);RVESFNYDWYNV (SEQ ID NO: 27); RVESFDFDWYNI (SEQ ID NO: 28); RINSFDYDWYNV(SEQ ID NO: 29); TVNSFDYDWYNV (SEQ ID NO: 30); KVNSFDYDWYNV (SEQ ID NO:31); TVHSFDYDWYNV (SEQ ID NO: 32); SVHSWDYDWYNV (SEQ ID NO: 33);SVHSYDFDWYNV (SEQ ID NO: 34); TLQAFNYEWYQL (SEQ ID NO: 35); KYETFEYGWYNI(SEQ ID NO: 36); HGDSFQYEWYNL (SEQ ID NO: 37); SVHSFDWDWYNV (SEQ ID NO:38); SVHSFDYDYYNV (SEQ ID NO: 39); SVHSFDYDFYNV (SEQ ID NO: 40);SVHSFDYDWFNV (SEQ ID NO: 41); SVHSFDYDWWNV (SEQ ID NO: 42); IFNPFDYDWYNV(SEQ ID NO: 43); QWHSFDYDWYNV (SEQ ID NO: 44) or DVHPFDYDWYNV (SEQ IDNO: 45).

The present invention also provides a method of dispersing a biofilm ordetaching biofilm formation from a surface, the method comprisingtreating water with or coating said surface with a compositioncomprising a protein or peptide, wherein the composition is capable ofdetaching a single cell organism from a surface or from other singlecell organisms. In some embodiments, the method comprises a peptideconsisting of amino acids X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ IDNO: 1), wherein X¹ is S, N, I, V, R, K, Q or L; X² is V, I, A, N, L orQ; X³ is P; X⁴ is Y, F or W; X⁵ is D, N, Q or E; X⁶ is Y, F, R, W, V, H,L, K or I; X⁷ is N, S, G, D, H, E, Q or I; X⁸ is W, L, F, M S, T, R, A,G, V, P, Y, I or K; X⁹ is Y, N, F, K, L, R, I, V, W or Q; X¹⁰ is S, K,N, T, E, R, L, Q, I, V, D, or K; X¹¹ is N, E, D, Q, S, A or I; and X¹²is W, R, V, L, I, K, F or E, wherein the peptide is not SVPYDYNWYSNW(SEQ ID NO: 2). In other embodiments, the peptide consists of aminoacids X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 3), wherein X¹is S, N, T, K, R, H, E, I, Q or D; X² is V, I, L, Y, G, F or W; X³ is H,N, Q, E, D or S; X⁴ is S, P, A or T; X⁵ is F, W or Y; X⁶ is D, N, E orQ; X⁷ is Y, F or W; X⁸ is D, G or E; X⁹ is W, F or Y; X¹⁰ is Y, F or W;X¹¹ is N or Q; and X¹² is V, I or L, wherein the peptide is notSVHSFDYDWYNV (SEQ ID NO: 4).

In specific embodiments, the peptide is SVPFDYNLYSNW (SEQ ID NO: 5);SAPYNFNFYSNW (SEQ ID NO: 6); NIPFNFSLNKER (SEQ ID NO: 7); SVPYQYNWYSNW(SEQ ID NO: 8); SVPWEYNFYSNW (SEQ ID NO: 9); RIPYDRGMIVNV (SEQ ID NO:10); KVPYDWDSVINL (SEQ ID NO: 11); QLPYDVHTYNDW (SEQ ID NO: 12);LAPYDHNRYTQW (SEQ ID NO: 13); SNPYDLEAYENW (SEQ ID NO: 14); SVPYDYQGYRNI(SEQ ID NO: 15); SVPYDYNVYLNK (SEQ ID NO: 16); IQPYDKNYFQNF (SEQ ID NO:17); VVPYDINIKDNW (SEQ ID NO: 18); SVPYDYNPYSNW (SEQ ID NO: 19);SVPYDYNKLKNW (SEQ ID NO: 20); SVPYDYNWRSSW (SEQ ID NO: 21); SVPYDYNWWSAW(SEQ ID NO: 22); SVPYDYNWQSNW (SEQ ID NO: 23); ELSSFNFDWYNV (SEQ ID NO:24); RYSSFDYDWYNV (SEQ ID NO: 25); NVHSFDYDWYNV (SEQ ID NO: 26);RVESFNYDWYNV (SEQ ID NO: 27); RVESFDFDWYNI (SEQ ID NO: 28); RINSFDYDWYNV(SEQ ID NO: 29); TVNSFDYDWYNV (SEQ ID NO: 30); KVNSFDYDWYNV (SEQ ID NO:31); TVHSFDYDWYNV (SEQ ID NO: 32); SVHSWDYDWYNV (SEQ ID NO: 33);SVHSYDFDWYNV (SEQ ID NO: 34); TLQAFNYEWYQL (SEQ ID NO: 35); KYETFEYGWYNI(SEQ ID NO: 36); HGDSFQYEWYNL (SEQ ID NO: 37); SVHSFDWDWYNV (SEQ ID NO:38); SVHSFDYDYYNV (SEQ ID NO: 39); SVHSFDYDFYNV (SEQ ID NO: 40);SVHSFDYDWFNV (SEQ ID NO: 41); SVHSFDYDWWNV (SEQ ID NO: 42); IFNPFDYDWYNV(SEQ ID NO: 43); QWHSFDYDWYNV (SEQ ID NO: 44) or DVHPFDYDWYNV (SEQ IDNO: 45).

The present invention also provides a method of treating a disease,comprising administering a peptide consisting of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO 1), wherein X¹ is S,N, I, V, R, K, Q or L; X² is V, I, A, N, L or Q; X³ is P; X⁴ is Y, F orW; X⁵ is D, N, Q or E; X⁶ is Y, F, R, W, V, H, L, K or I; X⁷ is N, S, G,D, H, E, Q or I; X⁸ is W, L, F, M S, T, R, A, G, V, P, Y, I or K; X⁹ isY, N, F, K, L, R, I, V, W or Q; X¹⁰ is S, K, N, T, E, R, L, Q, I, V, D,or K; X¹¹ is N, E, D, Q, S, A or I; and X¹² is W, R, V, L, I, K, F or E,wherein the peptide is not SVPYDYNWYSNW (SEQ ID NO: 2). In otherembodiments, the peptide consists of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 3), wherein X¹ is S,N, T, K, R, H, E, I, Q or D; X² is V, I, L, Y, G, F or W; X³ is H, N, Q,E, D or S; X⁴ is S, P. A or T; X⁵ is F, W or Y; X⁶ is D, N, E or Q; X⁷is Y, F or W; X⁸ is D, G or E; X⁹ is W, F or Y; X¹⁰ is Y, F or W; X¹¹ isN or Q; and X¹² is V, I or L, wherein the peptide is not SVHSFDYDWYNV(SEQ ID NO: 4).

In specific embodiments, the peptide is SVPFDYNLYSNW (SEQ ID NO: 5);SAPYNFNFYSNW (SEQ ID NO: 6); NIPFNFSLNKER (SEQ ID NO: 7); SVPYQYNWYSNW(SEQ ID NO: 8); SVPWEYNFYSNW (SEQ ID NO: 9); RIPYDRGMIVNV (SEQ ID NO:10); KVPYDWDSVINL (SEQ ID NO: 11); QLPYDVHTYNDW (SEQ ID NO: 12);LAPYDHNRYTQW (SEQ ID NO: 13); SNPYDLEAYENW (SEQ ID NO: 14); SVPYDYQGYRNI(SEQ ID NO: 15); SVPYDYNVYLNK (SEQ ID NO: 16); IQPYDKNYFQNF (SEQ ID NO:17); VVPYDINIKDNW (SEQ ID NO: 18); SVPYDYNPYSNW (SEQ ID NO: 19);SVPYDYNKLKNW (SEQ ID NO: 20); SVPYDYNWRSSW (SEQ ID NO: 21); SVPYDYNWWSAW(SEQ ID NO: 22); SVPYDYNWQSNW (SEQ ID NO: 23); ELSSFNFDWYNV (SEQ ID NO:24); RYSSFDYDWYNV (SEQ ID NO: 25); NVHSFDYDWYNV (SEQ ID NO: 26);RVESFNYDWYNV (SEQ ID NO: 27); RVESFDFDWYNI (SEQ ID NO: 28); RINSFDYDWYNV(SEQ ID NO: 29); TVNSFDYDWYNV (SEQ ID NO: 30); KVNSFDYDWYNV (SEQ ID NO:31); TVHSFDYDWYNV (SEQ ID NO: 32); SVHSWDYDWYNV (SEQ ID NO: 33);SVHSYDFDWYNV (SEQ ID NO: 34); TLQAFNYEWYQL (SEQ ID NO: 35); KYETFEYGWYNI(SEQ ID NO: 36); HGDSFQYEWYNL (SEQ ID NO: 37); SVHSFDWDWYNV (SEQ ID NO:38); SVHSFDYDYYNV (SEQ ID NO: 39); SVHSFDYDFYNV (SEQ ID NO: 40);SVHSFDYDWFNV (SEQ ID NO: 41); SVHSFDYDWWNV (SEQ ID NO: 42); IFNPFDYDWYNV(SEQ ID NO: 43); QWHSFDYDWYNV (SEQ ID NO: 44) or DVHPFDYDWYNV (SEQ IDNO: 45).

In some embodiments, the disease is autoimmune, inflammatory ordegenerative disease. In some embodiments, the disease is Alzheimer'sDisease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the detachment of Pseudomonas aeruginosa biofilm from awell in a 96-well culture plate after incubating the microorganismovernight to create the biofilm and subsequently incubating the biofilmwith Eqt2Z-cyc peptide overnight.

FIG. 2A shows the detachment of Pseudomonas aeruginosa biofilm from awell after incubating the microorganism for 2 hours to create thebiofilm and subsequently incubating the biofilm with differentconcentrations of Eqt2Z-cyc peptide and Abac10 negative controlovernight.

FIG. 2B shows the detachment of Pseudomonas aeruginosa biofilm from awell after incubating the microorganism for 2 hours to create thebiofilm and subsequently incubating the biofilm with differentconcentrations of Eqt2Z-cyc peptide and Abac10 negative control for 24hours.

FIG. 3 shows the detachment of Pseudomonas aeruginosa biofilm from awell after incubating the microorganism for 2 hours to create thebiofilm and subsequently incubating the biofilm with grZ14s-nvcycpeptide overnight.

FIG. 4 shows the detachment of Pseudomonas aeruginosa biofilm from awell after incubating the microorganism for 24 hours to create thebiofilm and subsequently incubating the biofilm with Eqt2-cyc peptidefor 24 hours.

FIG. 5 shows the detachment of Pseudomonas aeruginosa biofilm from awell after incubating the microorganism for 24 hours to create thebiofilm and subsequently incubating the biofilm with grZ14s-nvcycpeptide for 24 hours.

FIG. 6 shows the detachment of Pseudomonas aeruginosa biofilm from awell after incubating the microorganism for 24 hours to create thebiofilm and subsequently incubating the biofilm with Physco-cyc peptideovernight.

FIG. 7 shows the enhancement of Imipenem activity with grZ14s-nvCyc.

FIG. 8 shows the enhancement of Ampicillin activity with grZ14s-nvCyc.

FIG. 9 shows the enhancement of Vancomycin activity with grZ14s-nvCyc.

FIG. 10 shows the enhancement of Amphotericin activity withgrZ14s-nvCyc.

FIG. 11 shows the enhancement of Fluconazole activity with grZ14s-nvCyc

FIG. 12 shows the enhancement of Kanamycin activity with grZ14s-nvCyc

FIG. 13A shows the prevention of Pseudomonas aeruginosa adherence byvarious peptides.

FIG. 13B shows the detachment of Pseudomonas aeruginosa adherence byvarious peptides.

FIG. 14A shows the prevention of Staphylococcus aureus adherence byvarious peptides.

FIG. 14B shows the detachment of Staphylococcus aureus adherence byvarious peptides.

FIG. 15A shows the prevention of Candida albicans adherence by variouspeptides.

FIG. 15B shows the detachment of Candida albicans adherence by variouspeptides.

FIG. 16A shows the prevention of Escherichia coli adherence by variouspeptides.

FIG. 16B shows the detachment of Escherichia coli adherence by variouspeptides.

FIG. 17 shows the prevention of adhesion of Pseudomonas aeruginosa byvarious modifications of Eqt2Z-Cyc.

FIG. 18 shows Congo Red staining of Eqt2Z-Cyc incubated with Pseudomonasaeruginosa.

FIG. 19 shows Congo Red staining of grZ14s-nvCyc incubated withPseudomonas aeruginosa.

FIG. 20 shows Trypan Blue staining of grZ14s-nvCyc incubated withPseudomonas aeruginosa

FIG. 21 shows Congo Red staining of grZ14s-nvCyc incubated with Candidaalbicans.

FIG. 22 shows Congo Red staining of Eqt2Z-Cyc incubated with Candidaalbicans.

FIG. 23 shows Congo Red staining of grZ14s-nvCyc incubated withStaphylococcus aureus.

DETAIL DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to compositions and methods comprising aprotein or a peptide which has one or more effects relating to detachinga single cell organism from a surface or other single cell organisms,especially where the organism is in a biofilm.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are provided for theconvenience of the reader. All the information contained therein isincorporated herein by reference.

DEFINITIONS

As used herein, the term “isolated” refers to a composition that hasbeen removed from its in vivo location. Preferably the isolatedcompositions of the present invention are substantially free from othersubstances (e.g., other proteins that do not comprise anti-adhesiveeffects) that are present in their in vivo location (i.e., purified orsemi-purified). Isolated proteins and peptides may optionally besynthetic or obtained from natural sources, including optionally bybeing expressed in vivo using genetic engineering techniques.

As used herein the phrase “single cell organism” refers to a unicellularorganism also termed a microorganism or a microbe. The single cellorganism of the present invention can be a eukaryotic single cellorganism (e.g., protozoa or fungi for example yeast) or a prokaryoticsingle cell organism (e.g., bacteria or archaea). The single cellorganisms of the present invention may be in any cellular environment,such as for example, in a biofilm, as isolated cells or as a cellsuspension.

The term “Gram-positive bacteria” as used herein refers to bacteriacharacterized by having as part of their cell wall structurepeptidoglycan as well as polysaccharides and/or teichoic acids and arecharacterized by their blue-violet color reaction in the Gram-stainingprocedure. Representative Gram-positive bacteria include: Actinomycesspp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum,Clostridium perfringens, Clostridium spp., Clostridium tetani,Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcusfaecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae,Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum,Leuconostoc spp., Mycobacterium abscessus, Mycobacterium avium complex,Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacteriumhaemophilium, Mycobacterium kansasii, Mycobacterium leprae,Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacteriumsmegmatis, Mycobacterium terrae, Mycobacterium tuberculosis,Mycobacterium ulcerans, Nocardia spp., Peptococcus niger,Peptostreptococcus spp., Proprionibacterium spp., Sarcina lutea,Staphylococcus aureus, Staphylococcus auricularis, Staphylococcuscapitis, Staphylococcus cohnii, Staphylococcus epidermidis,Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcuslugdanensis, Staphylococcus saccharolyticus, Staphylococcussaprophyticus, Staphylococcus schleiferi, Staphylococcus similans,Staphylococcus warneri, Staphylococcus xylosus, Streptococcus agalactiae(group B streptococcus), Streptococcus anginosus, Streptococcus bovis,Streptococcus canis, Streptococcus equi, Streptococcus milleri,Streptococcus mitior, Streptococcus mutans, Streptococcus pneumoniae,Streptococcus pyogenes (group A streptococcus), Streptococcussalivarius, Streptococcus sanguis.

The term “Gram-negative bacteria” as used herein refer to bacteriacharacterized by the presence of a double membrane surrounding eachbacterial cell. Representative Gram-negative bacteria includeAcinetobacter calcoaceticus, Acinetobacter baumannii, Actinobacillusactinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xylosoxidans,Bacteroides, Bacteroides fragilis, Bartonella bacilliformis, Bordetellaspp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp.,Campylobacter spp., Chalmydia pneumoniae, Chlamydia psittaci, Chlamydiatrachomatis, Chromobacterium violaceum, Citrobacter spp., Eikenellacorrodens, Enterobacter aerogenes, Escherichia coli, Flavobacteriummeningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilusspp., Helicobacter pylori, Klebsiella pneumoniae, Klebsiella spp.,Legionella spp., Leptospira spp., Moraxella catarrhalis, Morganellamorganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseriameningitidis, Pasteurella multocida, Plesiomonas shigelloides,Prevotella spp., Proteus spp., Providencia rettgeri, Pseudomonasaeruginosa, Pseudomonas spp., Rickettsia prowazekii, Rickettsiarickettsii, Rochalimaea spp., Salmonella spp., Salmonella typhi,Serratia marcescens, Shigella spp., Shigella sonnei, Treponema carateum,Treponema pallidum, Treponema pallidum endemicum, Treponema pertenue,Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersiniaenterocolitica, Yersinia pestis.

The term “fungi” as used herein refers to the heterotrophic organismscharacterized by the presence of a chitinous cell wall, and in themajority of species, filamentous growth as multicellular hyphae.Representative fungi whose adhesion may be prevented according to themethod of the present invention include Candida albicans, Saccharomycescerevisiae, Candida glabrata, Candida parapsilosis and Candidadubliniensis.

As used herein the term “biofilm” refers to an extracellular matrix inwhich single cell organisms can be dispersed and/or form colonies. Thebiofilm typically is made of polysaccharides and other macromolecules.

As used herein, the term “detach” refers to removing a single cellorganism, in vitro or in vivo, from a surface to which the cell isadhered (e.g., by reducing the rate of growth on a surface) or removingthe cell from other single cell organisms to which they are adhered.Preferably, the compositions of the present invention detach cells fromadherence by as much as 10%, more preferably by 20%, more preferably by30%, more preferably by 40%, more preferably by 50%, more preferably by60%, more preferably by 70%, more preferably by 80%, more preferably by90% and most preferably by 100% as measured by an adhesion assay.Exemplary detachment assays are described herein below and in theExamples section that follows.

“Detachment” of a biofilm occurs when a single or cluster of cellorganisms in the biofilm detaches from a surface and “dispersion” of abiofilm occurs when single cell organisms in a biofilm detach from eachother.

As used herein the term “contacting” refers to the positioning of thecompositions of the present invention such that they are in direct orindirect contact with the adhesive single cell organisms in such a waythat the active agent comprised within is able to detach of cellstherefrom. Thus, the present invention contemplates both applying thecompositions of the present invention to a desirable surface and/ordirectly to the adhesive cells. Contacting the compositions with asurface can be effected using any method known in the art includingspraying, spreading, wetting, immersing, dipping, painting, ultrasonicwelding, welding, bonding or adhering. The compositions of the presentinvention may be attached as monolayers or multiple layers.

A “peptidomimetic organic moiety” can optionally be substituted foramino acid residues in a peptide according to the present invention bothas conservative and as non-conservative substitutions. These moietiesare also termed “non-natural amino acids” and may optionally replaceamino acid residues, amino acids or act as spacer groups within thepeptides in lieu of deleted amino acids. The peptidomimetic organicmoieties optionally and preferably have steric, electronic orconfigurational properties similar to the replaced amino acid and suchpeptidomimetics are used to replace amino acids in the essentialpositions, and are considered conservative substitutions. However suchsimilarities are not necessarily required. The only restriction on theuse of peptidomimetics is that the composition at least substantiallyretains its physiological activity as compared to the native peptideaccording to the present invention.

As used herein the term “chemical modification”, when referring to apeptide according to the present invention, refers to a peptide where atleast one of its amino acid residues is modified either by naturalprocesses, such as processing or other post-translational modifications,or by chemical modification techniques which are well known in the art.Examples of the numerous known modifications typically include, but arenot limited to: acetylation, acylation, amidation, ADP-ribosylation,glycosylation, GPI anchor formation, covalent attachment of a lipid orlipid derivative, methylation, myristylation, pegylation, prenylation,phosphorylation, ubiquitination, or any similar process.

As used herein the term “medical device” refers to any implant,instrument, apparatus, implement, machine, device or any other similaror related object (including any component or accessory), which isintended for use in the diagnosis, treatment, cure or prevention ofdisease or other conditions. Such medical device is intended for use inman or other animals and is anticipated to affect the structure or anyfunction of the body. Such medical device does not achieve its primaryintended purposes through chemical action and is not dependent uponbeing metabolized for the achievement of its primary intended purposes.

As used herein the term “implant” refers to any object intended forplacement in a human body that is not a living tissue. The implant maybe temporary or permanent. An implant can be an article comprisingartificial components, such as catheters or pacemakers. Implants canalso include naturally derived objects that have been processed so thattheir living tissues have been devitalized. As an example, bone graftsthat have been processed so that their living cells are removed(acellularized), but so that their shape is retained to serve as atemplate for ingrowth of bone from a host. As another example, naturallyoccurring coral can be processed to yield hydroxyapatite preparationsthat can be applied to the body for certain orthopedic and dentaltherapies. The present invention therefore envisions coating medicaldevices with the compositions of the present invention to preventmicroorganism adherence thereto so as to reduce/eliminate any possiblecell aggregation and biofilm formation known to occur followingimplantation. Device-related infections usually result from theintroduction of microorganisms, primarily bacteria, during the deviceinsertion or implantation procedure, or from attachment of blood-borneorganisms to the newly inserted device and their subsequent propagationon its surface. Coating the medical device with the compositions of thepresent invention will therefore inhibit biofilm formation of one ormore microbial species, will prevent medical device related infections,and consequently will reduce the need of antibiotic treatment or removalof the medical device from the subject.

As used herein the term “anti-biofouling agents” refers to the compoundsused to protect underwater surfaces from attaching single cellorganisms. These single cell organisms include microorganism such asbacteria and fungi.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier,” which may be usedinterchangeably, refer to a carrier or a diluent that does not causesignificant irritation to an organism and does not abrogate thebiological activity and properties of the administered compound. Anadjuvant is included under these phrases.

As used herein the term “excipient” refers to an inert substance addedto a pharmaceutical composition to further facilitate administration ofan active ingredient.

As used herein, the phrase “sessile aquatic organism” refers to anaquatic organism which is not freely moving for at least some a part ofits life cycle. Aquatic sessile organisms are usually permanentlyattached to a solid substrate of some kind, such as to a rock or thehull of a ship due to physical anchorage to the substrate, or for anyother reason (e.g., stone fish). Exemplary sessile organisms include,but are not limited to, sessile cnidarians such as corals, sea anemones(e.g., Actinia equine and Aiptasia pulchella), sea pens, aquatic sessilelarva (e.g., jellyfish larva), tube dwelling anemones and hydroids(e.g., Chlorohydra viridissima and Hydra vulgaris). Exemplary fish thatmay be used according to this aspect of the present invention arepreferably those dwelling in shallow waters or those that hide at thebottom layer of the ocean, sometimes in holes or caves. Such fishinclude eel and catfish.

As used herein the term “subject in need thereof” refers to a mammal,preferably a human subject.

As used herein the term “treating” refers to curing, reversing,attenuating, alleviating, minimizing, suppressing or halting thedeleterious effects of a pathogen infection.

As used herein the phrase “pathogen infection” refers to any medicalcondition which is caused by a pathogenic organism. Examples of pathogeninfections include, but are not limited to, chronic infectious diseases,subacute infectious diseases, acute infectious diseases, viral diseases,bacterial diseases, protozoan diseases, parasitic diseases, fungaldiseases, mycoplasma diseases, archaea diseases and prion diseases.Pathogen infection can be caused by an organism capable of growing in oron a biofilm. Examples of pathogen infections caused by microbialbiofilms include native valve endocarditis (NVE), otitis media (OM),chronic bacterial prostatitis, cystic fibrosis (CF) and periodontitis.Additional pathogen infections that are not specifically attributed tobiofilms include, but are not limited to urinary infections, femalegenital tract infections and pneumonia. Infections due to implantationof medical devices include vascular catheter infections, arterialprosthetic infections, infections of prosthetic heart valves, prostheticjoint infections, infections of central nervous system shunts,orthopedic implant infections, pacemaker and defibrillator infections,hemodialysis and peritoneal dialysis infections, ocular infections,urinary tract infections, infections of the female genital tract,infections associated with endotracheal intubation and tracheostomy anddental infections.

As used herein the phrase “pathogenic organism” refers to any singlecell organism which is capable of causing disease, especially a livingmicroorganism such as a bacteria or fungi. Preferably the pathogenicorganism is capable of growing in or on a biofilm. Many commonpathogenic organisms exist in mammals (e.g. humans) as biofilms andcause disease. These include, but are not limited to, Mannheimiahaemolytica and Pasteurella multocida (causing pneumonia), Fusobacteriumnecrophorum (causing liver abscess), Staphylococcus aureus andPseudomonas aeruginosa (causing wound infections), Escherichia coli andSalmonella spp (causing enteritis), Staphylococcus aureus andStaphylococcus epidermidis (causing OM), and Streptococci sp.,Staphylococci sp., Candida, and Aspergillus sp. (causing NVE).

Applications

The present invention has many applications. One application is to use acomposition comprising the protein or peptide to disperse a biofilm ordetach a biofilm from a surface. Another application is to use thecomposition to combat systemic infections (e.g., nosocomial infections)in patient caused by microbial biofilms in vivo. Another application isto use the composition to disperse or detach a biofilm present in otherfields, including the food, agriculture, pharmaceutical, paint, water,shipping and engineering industries. Another application is to use thecomposition to disperse or detach a biofilm where coaggregation i.e.,aggregation of more than one species that create a single clusteroccurs. The foregoing applications are not limiting and otherapplications are appropriate in which a composition can be used todetach a microorganism from a surface or from other microorganisms.Another application is to use the composition to treat disease,including autoimmune, inflammatory diseases and degenerative diseasessuch as Alzheimer's Disease. These diseases are caused by cellaggregation or clustering and the use of the inventive peptides toprevent or dissociate cell aggregates can lead to alleviation of thedisease. Another application is to use the composition to treatrestenosis, including restenosis caused by implanted medical stents.Another application is to use the composition to treat blood cellclustering, including white blood cells.

Exemplary bacterial cells that can be detached from surfaces and fromeach other according to the method of the present invention include grampositive bacteria and gram negative bacteria.

Exemplary surfaces upon which single cell organisms can attach and whichare contemplated by the present invention include fabrics, fibers,foams, films, concretes, masonries, glass, metals, plastics, polymers,and like.

Exemplary devices whose surfaces are susceptible to biofilm formationand which are contemplated by the present invention include, but are notlimited to, vessel hulls, automobile surfaces, air plane surfaces,membranes, filters, and industrial equipment. The surface may also becomprised in medical devices, instruments, and implants.

Examples of such medical devices, instruments, and implants include anyobject that is capable of being implanted temporarily or permanentlyinto a mammalian organism, such as a human. Representative medicaldevices, instruments, and implants that may be used according to thepresent invention include, for example, central venous catheters,urinary catheters, endotracheal tubes, mechanical heart valves,pacemakers, vascular grafts, stents and prosthetic joints.

Medical devices that may be coated according to the teachings of thepresent invention include, but are not limited to, artificial bloodvessels, catheters and other devices for the removal or delivery offluids to patients, artificial hearts, artificial kidneys, orthopedicpins, prosthetic joints, plates and implants; catheters and other tubes(including urological and biliary tubes, endotracheal tubes,peripherally insertable central venous catheters, dialysis catheters,long term tunneled central venous catheters, peripheral venouscatheters, short term central venous catheters, arterial catheters,pulmonary catheters, Swan-Ganz catheters, urinary catheters, peritonealcatheters), urinary devices (including long term urinary devices, tissuebonding urinary devices, artificial urinary sphincters, urinarydilators), shunts (including ventricular or arterio-venous shunts);prostheses (including breast implants, penile prostheses, vasculargrafting prostheses, aneurysm repair devices, mechanical heart valves,artificial joints, artificial larynxes, otological implants),anastomotic devices, vascular catheter ports, vascular stents, clamps,embolic devices, wound drain tubes, ocular lenses, dental implants,hydrocephalus shunts, pacemakers and implantable defibrillators,needleless connectors, voice prostheses and the like. Another possibleapplication of the compositions of the present invention is the coatingof surfaces found in the medical and dental environment. Such surfacesinclude the inner and outer aspects of various instruments and devices,whether disposable or intended for repeated uses. Such surfaces includethe entire spectrum of articles adapted for medical use, includingwithout limitation, scalpels, needles, scissors and other devices usedin invasive surgical, therapeutic or diagnostic procedures; bloodfilters. Other examples will be readily apparent to practitioners inthese arts.

Surfaces found in the medical environment also include the inner andouter aspects of pieces of medical equipment, medical gear worn orcarried by personnel in the health care setting. Such surfaces caninclude surfaces intended as biological barriers to infectious organismsin medical settings, such as gloves, aprons and face shields. Commonlyused materials for biological barriers are thermoplastic or polymericmaterials such as polyethylene, dacron, nylon, polyesters,polytetrafluoroethylene, polyurethane, latex, silicone and vinyl. Othersurfaces can include counter tops and fixtures in areas used for medicalprocedures or for preparing medical apparatus, tubes and canisters usedin respiratory treatments, including the administration of oxygen, ofsolubilized drugs in nebulizers and of anesthetic agents. Other suchsurfaces can include handles and cables for medical or dental equipmentnot intended to be sterile. Additionally, such surfaces can includethose non-sterile external surfaces of tubes and other apparatus foundin areas where blood or body fluids or other hazardous biomaterials arecommonly encountered. The compositions of the present invention can beused on the surface of or within these medical devices to provide longterm protection against colonization by single cell organisms and reducethe incidence of device-related infections. These compositions can alsobe incorporated in combination with an anti-microbial agent (e.g.,antibiotic agent) into coatings for medical devices. Such a combinationwill sufficiently kill or inhibit the initial colonizing bacteria andprevent device-related infections as long as the substance is presentedin an inhibitory concentration at the device-microbe interface.

The compositions of the present invention can be directly incorporatedinto the polymeric matrix of the medical device at the polymer synthesisstage or at the device manufacture stage. The compositions can also becovalently attached to the medical device polymer. These and many othermethods of coating medical devices are evident to one of ordinary skillin the art.

Additional surfaces that can be treated according to the teachings ofthe present invention include the inner and outer aspects of thosearticles involved in water purification, water storage and waterdelivery, and those articles involved in food processing. Thus thepresent invention envisions coating a solid surface of a food orbeverage container to extend the shelf life of its contents.

Surfaces related to health can also include the inner and outer aspectsof those household articles involved in providing for nutrition,sanitation or disease prevention. Thus, the compositions of the presentinvention can be used for removal of disease-causing microorganisms fromexternal surfaces. These can include, for example food processingequipment for home use, materials for infant care, tampons, soap,detergents, health and skincare products, household cleaners and toiletbowls.

The surface may be also be laboratory articles including, but notlimited to, microscopic slide, a culturing hood, a Petri dish or anyother suitable type of tissue culture vessel or container known in theart.

The inventors of this application also envision the use of thecompositions of the present invention as anti-Biofouling agents.

Underwater surfaces include any water immersed surface, includingships'/boats's hulls (i.e., the body or frame of a ship or boat),submergence vehicles, navigational aids, screens, nets, constructions,floating or emplaced offshore platforms (e.g., docks), buoys, signalingequipment and articles which come into contact with sea water or saltywater. Other underwater surfaces include structures exposed to sea waterincluding pilings, marine markers, undersea conveyances like cabling andpipes, fishing nets, bulkheads, cooling towers, and any device orstructure that operates submerged.

The compositions of the present invention can be incorporated intomarine coatings to limit undesirable marine biofouling. Thus, theanti-biofouling agents of the present invention can be formulated so asnot to contain toxic materials (such as heavy metals), and still retaintheir efficacy. The anti-biofouling paint of the present invention mayfurther contain binders(s), pigment(s), solvent(s) and additive(s).

Examples of solvents that may be used include aromatic hydrocarbons suchas xylene and toluene; aliphatic hydrocarbons such as hexane andheptane, esters such as ethyl acetate and butyl acetate; amides such asN-methylpyrrolidone and N,N-dimethylformamide; alcohols such asisopropyl alcohol and butyl alcohol; ethers such as dioxane, THF anddiethyl ether; and ketones such as methyl ethyl ketone, methyl isobutylketone and methyl isoamyl ketone. The solvents may be used alone or incombination thereof.

Examples of binders that may be used include alkyd resin, acrylic orvinyl emulsions, polyurethane resins, epoxy resins, silicone basedresins, acrylic resins, inorganic silicate based resins, vinyl resins,particularly a vinyl chloride/vinyl acetate copolymer, and rosin.Examples of pigments that may be used include titanium dioxide, cuprousoxide, iron oxide, talc, aluminum flakes, mica flakes, ferric oxide,cuprous thiocyanate, zinc oxide, cupric acetate meta-arsenate, zincchromate, zinc dimethyl dithiocarbamate, zinc ethylenebis(dithiocarbamate) and zinc diethyl dithiocarbamate.

Examples of additives that may be incorporated into the coatingcomposition include dehumidifiers, wetting/dispersing agents,anti-settling agents, anti-skinning agents, drying/curing agents,anti-marring agents and additives ordinarily employed in coatingcompositions as stabilizers and anti-foaming agents. Additionally, anyantibiotic which is relatively insoluble in seawater can be used with ananti-biofouling marine paint.

Methods of preparing marine anti-biofouling paints are explained indetail in U.S. Pat. No. 4,678,512; U.S. Pat. No. 4,286,988; U.S. Pat.No. 4,675,051; U.S. Pat. No. 4,865,909; and U.S. Pat. No. 5,143,545.

The compositions of the present invention may also be used for providingantibacterial properties in cosmetics, to prevent spoiling of theproduct.

The compositions may further be used to provide an antibacterial effectto the mouth, teeth and gums, such as by incorporation in a toothpaste,mouthwash, or chewing gum. Taken together the present teachings portraya wide range of novel anti-adhesive agents isolated from organisms suchas aquatic organisms and moss. The broad spectrum of the detachmenteffects of these anti-adhesive agents (e.g., removing adhesion of grampositive and gram negative bacteria) together with their ability toeffect the initial, vulnerable stages of microbial biofilm formation,makes these agents prime candidates as anti-biofilm agents. Moreover,the anti-adhesive agents described herein are clonable enablingmodifications and mass production thereof. In addition their stability(i.e., resistance to environmental conditions) makes these agentssuitable for a diverse array of applications.

It will be appreciated that treatment of infectious diseases accordingto the present invention may be combined with other treatment methodsknown in the art (i.e., combination therapy). These include, but are notlimited to, antimicrobial agents such as penicillins, cephalosporins,carbapenems, aminoglycosides, macrolides, lincomycins, tetracyclines,chloramphenicol, and griseofulvin. Suitable routes of administrationmay, for example, include oral, rectal, transmucosal, especiallytransnasal, intestinal, or parenteral delivery, including intramuscular,subcutaneous, and intramedullary injections, as well as intrathecal,direct intraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, or lyophilizing processes. Pharmaceuticalcompositions for use in accordance with the present invention thus maybe formulated in conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries, whichfacilitate processing of the active ingredients into preparations thatcan be used pharmaceutically. Proper formulation is dependent upon theroute of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For topical administration, the compositions of the present inventionmay be formulated as a gel, a cream, a wash, a rinse or a spray.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries as desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, and sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents, such ascross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses. Pharmaceuticalcompositions that can be used orally include push-fit capsules made ofgelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules maycontain the active ingredients in admixture with filler such as lactose,binders such as starches, lubricants such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active ingredientsmay be dissolved or suspended in suitable liquids, such as fatty oils,liquid paraffin, or liquid polyethylene glycols. In addition,stabilizers may be added. All formulations for oral administrationshould be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane, or carbon dioxide. In the case of apressurized aerosol, the dosage may be determined by providing a valveto deliver a metered amount. Capsules and cartridges of, for example,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base, such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with, optionally, anadded preservative. The compositions may be suspensions, solutions, oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing, and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water-based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters such as ethyl oleate, triglycerides, orliposomes. Aqueous injection suspensions may contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.

Optionally, the suspension may also contain suitable stabilizers oragents that increase the solubility of the active ingredients, to allowfor the preparation of highly concentrated solutions. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g., a sterile, pyrogen-free, water-based solution, beforeuse.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, for example, conventional suppository bases such as cocoabutter or other glycerides. Pharmaceutical compositions suitable for usein the context of the present invention include compositions wherein theactive ingredients are contained in an amount effective to achieve theintended purpose. More specifically, a “therapeutically effectiveamount” means an amount of active ingredients (e.g., an aquatic organismcomposition) effective to prevent, alleviate, or ameliorate symptoms ofa pathogenic infection (e.g., fever) or prolong the survival of thesubject being treated. Determination of a therapeutically effectiveamount is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the dosage orthe therapeutically effective amount can be estimated initially from invitro and cell culture assays. For example, a dose can be formulated inanimal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans. Toxicity and therapeutic efficacy of the active ingredientsdescribed herein can be determined by standard pharmaceutical proceduresin vitro, in cell cultures or experimental animals. The data obtainedfrom these in vitro and cell culture assays and animal studies can beused in formulating a range of dosage for use in human. The dosage mayvary depending upon the dosage form employed and the route ofadministration utilized. The exact formulation, route of administration,and dosage can be chosen by the individual physician in view of thepatient's condition. (See, e.g., Fingl, E. et al. (1975), “ThePharmacological Basis of Therapeutics,” Ch. 1, p. 1.) Dosage amount andadministration intervals may be adjusted individually to providesufficient plasma or brain levels of the active ingredient to induce orsuppress the biological effect (i.e., minimally effective concentration,MEC). The MEC will vary for each preparation, but can be estimated fromin vitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks, oruntil cure is effected or diminution of the disease state is achieved.The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA-approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser device may also be accompaniedby a notice in a form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration. Such notice, for example, may includelabeling approved by the U.S. Food and Drug Administration forprescription drugs or of an approved product insert. Compositionscomprising a preparation of the invention formulated in a‘pharmaceutically acceptable carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition, as further detailed above.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

Proteins and Peptides

The protein or peptide in the compositions can be obtained fromdifferent sources. Natural proteins and peptides can be found inanimals, including humans. Also, marine and fresh water plants andorganisms, including soft bodied water invertebrates, fish and mossproduce several factors that can prevent microbial colonization on theirbody surface since they lack specific immunity and are surrounded bybroad spectrum species of microbial organisms. The most “sensitive”organisms are invertebrates belong to the phylum cnidaria that includethe sea anemones, corals, jellyfish, hydroids, medusae, and sea fans.Such soft bodied organism, which lack physical protection such as scalesor shells, use proteins and secondary metabolites to protect themselvesfrom the microbial environment surrounding them.

Additionally, it has been previously reported that marine organisms(e.g., sponges) produce secondary metabolites that exhibit antibacterialand antifungal activities [Amade et al., supra]. Moreover, sea anemones(e.g., Actinia equina) have been shown to produce toxic, pore formingpeptides (i.e., equinatoxins), which lyse and kill eukaryotic cellssimilarly to other small antimicrobial peptides [Anderluh et al.,supra].

Although it is known in the art that the full length sequences ofvarious proteins are related to their cytolysic function, the specificpeptides responsible for the cytolysic effect have not been previouslyidentified.

Whilst reducing the present invention to practice, the present inventorsdiscovered that human proteins and aquatic sessile organisms compriseanti-biofilm properties. As is shown in the Examples section whichfollows, the present inventors have shown that proteins and peptidesfound in humans and sessile cnidarians (e.g., sea anemones) can causemicroorganisms to detach from surfaces and from each other (FIGS. 1-6).These proteins and peptides were not bactericidal and did not affectbacterial growth.

Taken together the present teachings portray a wide range of novelanti-adhesive agents derived from human and aquatic sessile organisms,in particular from sessile cnidarians. The broad spectrum of theanti-biofilm effects of these agents makes these agents prime candidatesas anti-biofilm agents. Moreover, the anti-adhesive agents describedherein are clonable enabling modifications and mass production thereof.In addition their stability (i.e., resistance to environmentalconditions) makes these agents suitable for a diverse array ofapplications.

The present inventors have demonstrated that several active fractionsobtained from sea anemones using liquid chromatography separations showa high level of prevention of microbial adherence to abiotic surfaces.The sea anemone includes 46 families that can be found in water sourcesaround the world. Most sea anemones are sessile, with a specialized footused to anchor them in soft substrates, or attach themselves to rocksand corals. The anti-adhesive activity was demonstrated with severalspecies of sea anemone belonging to different genera: Actinia equine,Aiptasia and Ammonia. The N terminus region of anemone cytotoxin hasbeen shown to be involved in the cytotoxic effect [Ref: Kristan K,Podlesek Z, Hojnik V, Gutierrez-Aguirre I₅ Guncar G, Turk D,Gonzalez-Manas J M, Lakey J H, Macek P, Anderluh G (2004): Poreformation by equinatoxin, an eukaryotic pore-forming toxin, requires aflexible N-terminal region and a stable beta sandwich. J Biol Chem.279(45):46509-46517]. A protein having some resemblance to the Cterminus region of anemone cytotoxin superfamily (pfam06369) (asuperfamily cluster is a set of conserved domain models, from one ormore source databases, that generate overlapping annotation on the sameprotein sequences. These models are assumed to represent evolutionarilyrelated domains and may be redundant with each other), which region isnot involved in cytotoxicity, has also been identified in fish by thepresent inventors. This protein has a highly conserved region, withunknown function, which is also a Trp-rich domain, and may be importantfor binding of the protein to lipid membrane. The present inventors havealso found this region in the moss Physcomitrella patens. The presentinventors therefore hypothesized that this region provides a peptidewhich is highly effective in dispersing biofilms or detaching biofilmsfrom surfaces, while being devoid of cytotoxic activity.

Based on bioinformatics analysis, it is believed that the protein haschanged in upper organisms (including in humans), at the highlyconserved region, to FDYDWY (SEQ ID NO: 46), that can be found inproteins, represented in the GenBank as GPCR 137b like, with size rangefrom 128aa-400aa. In humans, this peptide is part of the Gprotein-coupled receptor 137B (GENE ID: 7107 GPR137B) located at269-274. Based on UniProtKB/Swiss-Prot entry 060478, the region, whichstarts at 259 and ends at 292, is an extracellular region, whichsupports the theory that this peptide is the active part of the protein.Bioinformatics analysis of the ancient sea organism Ciona intestinalisidentified a 368 amino acid protein, similar to the G protein-coupledreceptor 137ba [GeneBank Accession number XP_002125109]. The regionsimilar to the anti adhesive peptide is SPLRCSELSSFNFDWYNVSDQADLVN (SEQID NO: 47). Based on this information, and the fact that Cionaintestinalis is also exposed to a large diversity of microorganisms, thepeptide FNFDWY (SEQ ID NO: 48) is also highly effective in dispersingbiofilms or detaching biofilms from surfaces, while being devoid ofcytotoxic activity.

The protein or peptide may be natural and isolated from any animal.Preferably, the animal is a vertebrate, such as, for example, a fish, anamphibian (including a frog, a toad, a newt or a salamander), a bird, areptile (such as a crocodilee, a lizard, a snake, a turtle, a tortoiseor a terrapin) or a mammal (including a human).

According to some embodiments, the peptide comprises part of a sequencecomprising up to about 30, up to about 40, or up to about 50 aminoacids.

The peptides of the present invention may optionally comprise at leasttwo sequences, connected by a linker of some type, such that theN-terminal of a first peptide sequence is connected to the C-terminal ofthe linker, and the C-terminal of a second peptide sequence is connectedto the N-terminal of the linker.

The peptides of the present invention can be cyclized (i.e., in cyclicform) and are indicated in this application as such with the term “cyc.”

In one embodiment, each peptide is modified with a cysteine at theC-terminal and a cysteine at the N-terminal, wherein the C- andN-terminals are S-S bridged. In specific embodiments, one or more of thepeptides are modified with a cysteine at the C-terminal and a cysteineat the N-terminal, wherein the C- and N-terminals are S-S bridged.

Exemplary peptides contain a domain which comprises at least one peptideand which is effective against adhesion of a single cell organism to asurface or to other single cell organisms. More preferably, the domainis included as part of a protein. Optionally and most preferably, thedomain exhibits anti-adhesive behavior, for example for the detachmentand/or treatment of a biofilm, but does not exhibit cytotoxic behavior.

The peptides may optionally be altered so as to form non-peptideanalogs, including but not limited to replacing one or more bonds withless labile bonds, cyclization and the like. Additionally oralternatively, a peptide may optionally be converted to a small moleculethrough computer modeling, as described for example in PCT ApplicationNo. WO/2007/147098, hereby incorporated by reference as if fully setforth herein.

Peptidomimetics may optionally be used to inhibit degradation of thepeptides by enzymatic or other degradative processes. Thepeptidomimetics can optionally and preferably be produced by organicsynthetic techniques. Non-limiting examples of suitable peptidomimeticsinclude D amino acids of the corresponding L amino acids, tetrazol(Zabrocki et al., J. Am. Chem. Soc. 110:5875 5880 (1988)); isosteres ofamide bonds (Jones et al, Tetrahedron Lett. 29: 3853 3856 (1988)); LL 3amino 2 propenidone 6 carboxylic acid (LL Acp) (Kemp et al., J. Org.Chem. 50:5834 5838 (1985)). Similar analogs are shown in Kemp et al.,Tetrahedron Lett. 29:5081 5082 (1988) as well as Kemp et al.,Tetrahedron Lett. 29:5057 5060 (1988), Kemp et al., Tetrahedron Lett.29:4935 4938 (1988) and Kemp et al., J. Org. Chem. 54:109 115 (1987).Other suitable but exemplary peptidomimetics are shown in Nagai andSato, Tetrahedron Lett. 26:647 650 (1985); Di Maio et al., J. Chem. Soc.Perkin Trans., 1687 (1985); Kahn et al., Tetrahedron Lett. 30:2317(1989); Olson et al., J. Am. Chem. Soc. 112:323 333 (1990); Garvey etal., J. Org. Chem. 56:436 (1990). Further suitable exemplarypeptidomimetics include hydroxy 1,2,3,4 tetrahydroisoquinoline 3carboxylate (Miyake et al., J. Takeda Res. Labs 43:53 76 (1989));1,2,3,4 tetrahydro-isoquinoline 3 carboxylate (Kazmierski et al., J. Am.Chem. Soc. 133:2275 2283 (1991)); histidine isoquinolone carboxylic acid(HIC) (Zechel et al., Int. J. Pep. Protein Res. 43 (1991)); (2S,3S)methyl phenylalanine, (2S,3R) methyl phenylalanine, (2R,3S) methylphenylalanine and (2R,3R) methyl phenylalanine (Kazmierski and Hruby,Tetrahedron Lett. (1991)).

Exemplary, illustrative but non-limiting non-natural amino acids includebeta-amino acids (beta3 and beta2), homo-amino acids, cyclic aminoacids, aromatic amino acids, Pro and Pyr derivatives, 3-substitutedAlanine derivatives, Glycine derivatives, ring-substituted Phe and TyrDerivatives, linear core amino acids or diamino acids. They areavailable from a variety of suppliers, such as Sigma-Aldrich (USA) forexample. In the present invention any part of a peptide may optionallybe chemically modified, i.e., changed by addition of functional groups.The modification may optionally be performed during synthesis of themolecule if a chemical synthetic process is followed, for example byadding a chemically modified amino acid. However, chemical modificationof an amino acid when it is already present in the molecule (“in situ”modification) is also possible.

The amino acid of any of the sequence regions of the molecule canoptionally be modified according to any one of the following exemplarytypes of modification (in the peptide conceptually viewed as “chemicallymodified”). Non-limiting exemplary types of modification includecarboxymethylation, acylation, phosphorylation, glycosylation or fattyacylation. Ether bonds can optionally be used to join the serine orthreonine hydroxyl to the hydroxyl of a sugar. Amide bonds canoptionally be used to join the glutamate or aspartate carboxyl groups toan amino group on a sugar (Garg and Jeanloz, Advances in CarbohydrateChemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang.Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal bonds canalso optionally be formed between amino acids and carbohydrates. Fattyacid acyl derivatives can optionally be made, for example, by acylationof a free amino group (e.g., lysine) (Toth et al., Peptides: Chemistry,Structure and Biology, Rivier and Marshal, eds., ESCOM Publ., Leiden,1078-1079 (1990)).

The compositions of the present invention may also be expressed in-vivousing genetic engineering techniques (e.g., using transgenic aquaticsessile organisms).

The compositions of the present invention may be devoid of cytotoxic orcytostatic activity—e.g., they are not bactericidal or bacteristatic.

The present inventors have characterized and isolated a natural peptidecomprising a sequence selected from the group consisting of YDYNWY (SEQID NO: 49), YDYNLY (SEQ ID NO: 50), FDYNFY (SEQ ID NO: 51), FDYNLY (SEQID NO: 52), WDYNLY (SEQ ID NO: 53), FDYNWY (SEQ ID NO: 54), YDWNLY (SEQID NO: 55), YDWHLY (SEQ ID NO: 56) and YDYSFY (SEQ ID NO: 57) havingeffective anti-cell aggregate (e.g., anti-biofilm) properties. Forexample, the peptide may comprise at least one of the followingsequences: LFSVPYDYNWYSNWW (SEQ ID NO: 58), LFSVPYDYNWYSNWW (SEQ ID NO:59), FSVPYDYNLYSNWW (SEQ ID NO: 60), MFSVPYDYNLYSNWV (SEQ ID NO: 61),MFSVPFDYNFYSNWW (SEQ ID NO: 62), LFSVPFDYNFYSNWW (SEQ ID NO: 63),MFSVPFDYNLYSNWW (SEQ ID NO: 64), MFSVPFDYNLYTNWW (SEQ ID NO: 65),MWSVPFDYNLYSNWW (SEQ ID NO: 66), MFSVPFDYNLYKNWL (SEQ ID NO: 67),LFSVPFDYNLYSNWW (SEQ ID NO: 68), LFSIPFDYNLYSNWW (SEQ ID NO: 69),MFSVPWDYNLYKNWL (SEQ ID NO: 70), MFSVPWDYNLYKNWF (SEQ ID NO: 71),MFSVPFFDYNWYSNWW (SEQ ID NO: 72), MASIPYDWNLYQSWA (SEQ ID NO: 73),MASIPYDWNLYSAWA (SEQ ID NO: 74) or MASIPYDWHLYNAWA (SEQ ID NO: 75), orcombinations thereof.

The present inventors have characterized and isolated a natural peptidecomprising the sequence selected from the group consisting of: FDYDWY(SEQ ID NO: 46), FNFDWY (SEQ ID NO: 48) and FDFDWY (SEQ ID NO: 76),having effective anti-cell aggregate (e.g., anti-biofilm) properties.For example, the peptide may comprise at least one of the followingsequences: SFDYDWY (SEQ ID NO: 77), SFDYDWYN (SEQ ID NO: 78), HSFDYDWYN(SEQ ID NO: 79), HSFDYDWYNV (SEQ ID NO: 80), VHSFDYDWYNV (SEQ ID NO:81), VHSFDYDWYNVS (SEQ ID NO: 82), SVHSFDYDWYNVS (SEQ ID NO: 83),SVHSFDYDWYNVSD (SEQ ID NO: 84), KSVHSFDYDWYNVSD (SEQ ID NO: 85),KSVHSFDYDWYNVSDQ (SEQ ID NO: 86), NKSVHSFDYDWYNVSDQ (SEQ ID NO: 87),NKSVHSFDYDWYNVSDQA (SEQ ID NO: 88), QNKSVHSFDYDWYNVSDQA (SEQ ID NO: 89),QNKSVHSFDYDWYNVSDQAD (SEQ ID NO: 90), SQNKSVHSFDYDWYNVSDQAD (SEQ ID NO:91), SQNKSVHSFDYDWYNVSDQADL (SEQ ID NO: 92), FSQNKSVHSFDYDWYNVSDQADL(SEQ ID NO: 93), FSQNKSVHSFDYDWYNVSDQADLK (SEQ ID NO: 94),SFSQNKSVHSFDYDWYNVSDQADLK (SEQ ID NO: 95), SFSQNKSVHSFDYDWYNVSDQADLKN(SEQ ID NO: 96), CSFSQNKSVHSFDYDWYNVSDQADLKN (SEQ ID NO: 97) orCSFSQNKSVHSFDYDWYNVSDQADLKNC (SEQ ID NO: 98), or combinations thereof.

The present inventors have characterized and isolated a natural peptidecomprising a sequence selected from the group consisting of SVPYDYNWYSNW(SEQ ID NO: 2), SFSQNKSVHSFDYDWYNVSDQADLKN (SEQ ID NO: 96) andSVHSFDYDWYNV (SEQ ID NO: 4).

An exemplary protein or peptide agent derived from an aquatic organismmay be used to detach a single cell organism from a surface or fromother single cell organisms is equinatoxin. Equinatoxins (i.e.,equinatoxins 1, 2 and 3) have pore forming toxins found in sea anemones(e.g., Actinia equina). Equinatoxins may be comprised in sea anemonecells or may be isolated therefrom. Any equinatoxin may be usedaccording to the teachings of the present invention for detachingmicroorganisms from a surface or from each other. One example is theEquinatoxin-2 precursor (GenBank accessionidentifier >gi|48428895|sp|P61914.1|ACTP2_ACTEQ (Equinatoxin II) (EqTII) (EqTII)):

(SEQ ID NO: 99) MSRLIIVFIVVTMICSATALPSKKIIDEDEEDEKRSADVAGAVIDGASLSFDILKTVLEALGNVKRKIAVGVDNESGKTWTALNTYFRSGTSDIVLPHKVPHGKALLYNGQKDRGPVATGAVGVLAYLMSDGNTLAVLFSVPYDYNWYSNWWNVRIYKGKRRADQRMYEELYYNLSPFRGDNGWHTRNLGYGLKSRGFMNSSGHAILEIHVSKA

Another exemplary protein derived from a sea anemone is Stichodactylahelianthus ((GenBank accessionidentifier >gi|2815496|sp|P07845.2|ACTP2_STOHE (Sticholysin II) (StnII)(Cytolysin St II) (Cytolysin III) (Cytotoxin)):

(SEQ ID NO: 100) ALAGTIIAGASLTFQVLDKVLEELGKVSRKIAVGIDNESGGTWTALNAYFRSGTTDVILPEFVPNTKALLYSGRKDTGPVATGAVAAFAYYMSSGNTLGVMFSVPFDYNWYSNWWDVKIYSGKRRADQGMYEDLYYGNPYRGDNGWHEKNLGYGLRMKGIMTSAGEAKMQIKISR

An exemplary protein derived from bony fish is Danio rerio ((GenBankaccession identifier >gi|125821212|ref|XP_001342650.1|PREDICTED (Daniorerio)):

(SEQ ID NO: 101) MTESAEAVAANVSSRRHATVEITNLTNNYCFLNPKVYLENGETSNPPQPTVRPLKTEVCTFSKSAAHATGSVGVLTYDLFERRRNDYTETLAIMFSVPWDYNLYKNWFAVGIYPKGKECDQALYKEMYYQKNQHGFVREEANGSGINFEGKNLDIRATMCPMGRAIVKVEVWDKLLSPMA QMDC

Another exemplary protein derived from bony fish is Tetradodonnigroviridis ((GenBank accessionidentifier >gi|47218822|emb|CAG02807.1|unnamed protein product (Teraodonnigroviridis)):

(SEQ ID NO: 102) MESAEAVAADVSRSRSVTIEISNLTKNYCLINPRVYLESGETYNPPQPTVRPLMTEVCTFSKSSGIPTGSVGVLTYELLERRSTMLPETLAIMFSVPYDYSFYNNWFAVGIYETGTKCNEGLYKQMYNEKKQAEHGFVREKANGSGINYVGGNLDIRATMNPLGKAIMKVEVWDAFFPFS E

An exemplary protein derived from moss is Physcomitrella patens((GenBank accession identifier >gi|16806023|ref|XP_001782104.1|predictedprotein (Physcomitrella patens subsp. patens)):

(SEQ ID NO: 103) MVVHLIAMGLRYSETIMKTARMAEAIIPAAELSIKTLQNIVEGITGVDRKIAIGFKNLTDYTLENLGVYFNSGSSDRSIAYKINAQEALLFSARKSDHTARGTVGTFSYYIQDEDKTVHVMWSVPFDYNLYSNWWNIAVVDGRQPPDSNVHDNLYNGSGGMPYPNKPDQYINNEQKGFHL FGSMTNNGQATIEVELKKA

An exemplary protein derived from birds is Gallus gallus (GenBankaccession identifier >gi|118129726|ref|XP_001231839.1|PREDICTED:hypothetical protein isoform (Gallus gallus)):

(SEQ ID NO: 104) MPPKEKKENDKPCNDNCQPKPQGKGVESLMKNIDVCRSVGLEIINRTRTVTLTDFRSYCFSGKIVTTLPFEIGPDSKGICIFAKTPYSLRGSVGTVVCKADTFFLAITFSNPYDYILYKIEFALEIFTEPNHLGNLGDVFSKMMKSKPYCGSSLFQRAVLESEHETLEVSKGSIRVQAKM SNNRKAILKVQVEDMDPPPYSKGM

An exemplary protein derived from a platypus is Ornithorhynchus anatinus(GenBank accessionidentifier >gi|149638239|ref|XP_001512702.1|PREDICTED: integral membraneprotein GPR137B-like (Ornithorhynchus anatinus)):

(SEQ ID NO: 105) MEGSPPGRPPGNDSLPPTLSPAVPPYVKLGLTSVYTAFYSLLFVFVYAQLWLVLHHRHRRLSYQTVFLFLCLLWAALRTVLFSFYFRDFLAANKLGPFGFWLLYCCPVCLQFFTLTLMNLYFSQVIFKAKSKFSPELLKYRLALYLASLVVSLVFLLVNLTCAVLVKTGTWERKVVVSVRVAINDTLFVLCAVSLSVCLYKISKMSLANIYLESKGSSVCQVTAIGVTVILLYASRACYNLFTLSFSRHGSSFDYDWYNVSDQADLKSQLGDAGYVVFGVVLFVWELLPTSLVVYFFRVRNPTKDPTNPRGVPSHAFSPRSYFFDNPRRYDSDDDLAWNVAPQGFQGSFAPDYYDWGQPS SSFTGHIGSLQQDSDLDNGKPSHA

An example of an applicable peptide derived from equinatoxin isSVPYDYNWYSNW (SEQ ID NO: 2), in either soluble form or conjugated topolyethylene glycol (PEG) and palmitic acid and capable of adhering to asurface. The soluble and conjugated form of the peptide in cyclic formand having a cysteine at each of the N- and C-terminal ends are asfollows:

Eqt2ZCyc (soluble aquatic peptide): (SEQ ID NO: 106) CSVPYDYNWYSNWCEqt2ZCyc-3PEG-Pal (adhesive aquatic peptide): (SEQ ID NO: 107)Pal--(miniPEG)3-CSVPYDYNWYSNWCPeptides based on a subset of equinatoxin sequences and variants thereofare also applicable. The chart below demonstrates specific 12-merpeptide embodiments, with applicable amino acids for each of the 12positions. Bolded amino acids correspond to amino acids not naturallyoccurring at the corresponding positions, but which provide for peptidesfalling within the scope of the present invention. In some embodiments,the peptide is not SVPYDYNWYSNW (SEQ ID NO: 2).

1 2 3 4 5 6 7 8 9 10 11 12 Equinatoxin S V P Y D Y N W Y S N W(SEQ ID NO: 1) N I F N F S L N K E R I A W Q R G F F N D V V N E W D M KT Q L R L V H S L E S I K Q H E T R R A K Q L Q R I L I F L K I A V Q EI G W I V Q V P D Y K I KAn exemplary peptide agent may be derived from a human. For example, apeptide derived from GPCR137b (GPR137B (TM7SF1)) may be used to detach asingle cell organism from a surface or from other single cell organisms.GPCR137b can be found in human kidney, heart, brain, and placentatissue. [Spangenberg et al., Genomics. 1998 Mar. 1; 48(2):178-85;Bjarnadottir et al., Genomics, 88(3): 263-273 (2006)]. PeptidesSFSQNKSVHSFDYDWYNVSDQADLKN (SEQ ID NO: 96) and SVHSFDYDWYNV (SEQ ID NO:4), as cyclic peptides with cysteine attached to both the C- andN-terminals of the peptides in either soluble form or conjugated topolyethylene glycol and palmitic acid and capable of adhering to asurface, are two examples of applicable peptides derived from humanGPCR137b:

grZ28Cyc (soluble long human peptide): (SEQ ID NO: 98)CSFSQNKSVHSFDYDWYNVSDQADLKNCgrZ28Cyc-3PEG-Pal (adhesive long human peptide): (SEQ ID NO: 108)Palmitoyl-(miniPEG)3-CSFSQNKSVHSFDYDWYNVSDQADLKNCgrZ14s-nvCyc (soluble short human peptide): (SEQ ID NO: 109)CSVHSFDYDWYNVC grZ14s-nvCyc-3PEG-Pal (adhesive short human peptide):(SEQ ID NO: 110) Pal--(miniPEG)3-CSVHSFDYDWYNVCPeptides based on a subset of GPCR137b sequences and variants thereofare also applicable. The chart below demonstrates specific 12-merpeptide embodiments, with applicable amino acids for each of the 12positions. Bolded amino acids correspond to amino acids not naturallyoccurring at the corresponding positions, but which provide for peptidesfalling within the scope of the present invention. In some embodiments,the peptide is not SVHSFDYDWYNV (SEQ ID NO: 4).

1 2 3 4 5 6 7 8 9 10 11 12 GPCR137B S V H S F D Y D W Y N V(SEQ ID NO: 3) N I N P W N F G F F Q I T L Q A Y E W E Y W L K Y E T Q RG D H F S E W I Q D

An exemplary peptide derived from Physcomitrella patens (moss) isSVPFDYNLYSNW (SEQ ID NO: 111). This same sequence can also be found inSelaginella moellendorffii (Genbank Accession No: XP_002963283), a typeof plant, and in the following sea anemones: Phyllodiscus semoni(Genbank Accession No: BAI70365); Heteractis crispa (Genbank AccessionNo: AAW47930); and Actineria villosa (Genbank Accession No: BAD74019).The cyclicized form of the peptide is as follows:

Physco-Cyc: (SEQ ID NO: 112) CSVPFDYNLYSNWC

EXAMPLES Example 1 Detaching Overnight Biofilm with Overnight Eqt2Z-cycIncubation

A biofilm was created by incubating Pseudomonas aeruginosa (ATCC 27853)overnight in wells at 37° C., allowing the microorganism to attach tothe well surface. Eqt2Z-cyc peptide [Custom Peptide Synthesis; Peptron,Korea] at different concentrations was subsequently added to the wellsand allowed to incubate overnight. The P.a. control was Pseudomonasaeruginosa alone. FIG. 1 demonstrates that Eqt2Z-cyc peptides caused fargreater microorganism detachment than in the P.a. control.

Example 2 Detaching 2-Hour Biofilm with Overnight Eqt2Z-cyc Incubation

A biofilm was created by culturing Pseudomonas aeruginosa (ATCC 27853)for 2 hours in a well at 37° C. Eqt2Z-cyc peptide at differentconcentrations was subsequently added to the well and allowed toincubate overnight. The Abac10-cyc peptide served as a negative control.The P.a. control was Pseudomonas aeruginosa alone. FIG. 2A demonstratesthat Eqt2Z-cyc peptides caused far greater microorganism detachment thanin the P.a. control or negative control.

Example 3 Detaching 2-Hour Biofilm with 24-Hour Eqt2Z-cyc Incubation

A biofilm was created by culturing Pseudomonas aeruginosa (ATCC 27853)for 2 hours in a well at 37° C. Eqt2Z-cyc peptide at differentconcentrations was subsequently added to the well and allowed toincubate for 24 hours. The Abac10-cyc peptide served as a negativecontrol. The P.a. control was Pseudomonas aeruginosa alone. FIG. 2Bdemonstrates that Eqt2Z-cyc peptides caused far greater microorganismdetachment than in the P.a. control or negative control. Staining wasperformed with crystal violet at OD 595 nm.

Example 4 Detaching 2-Hour Biofilm with 24-Hour grZ14s-nvCyc Incubation

A biofilm was created by culturing Pseudomonas aeruginosa (ATCC 27853)for 2 hours in a well at 37° C. grZ14s-nvCyc peptide [Custom PeptideSynthesis; Peptron, Korea] at different concentrations was subsequentlyadded to the well and allowed to incubate for 24 hours. The Abac10-cycpeptide served as a negative control. The P.a. control was Pseudomonasaeruginosa alone. FIG. 3 demonstrates that grZ14s-nvCyc peptides causedfar greater microorganism detachment than in the P.a. control ornegative control. Staining was performed with crystal violet at OD 595nm.

Example 5 Detaching 24-Hour Biofilm with 24-Hour Eqt2Z-cyc Incubation

A biofilm was created by culturing Pseudomonas aeruginosa (ATCC 27853)for 24 hours in a well at 37° C. Eqt2Z-cyc peptide at differentconcentrations was subsequently added to the well and allowed toincubate for 24 hours. The Abac10-cyc peptide served as a negativecontrol. The P.a. control was Pseudomonas aeruginosa alone. FIG. 4demonstrates that Eqt2Z-cyc peptides caused far greater microorganismdetachment than in the P.a. control or negative control. Staining wasperformed with crystal violet at OD 595 nm.

Example 6 Detaching 24-Hour Biofilm with 24-Hour grZ14s-nvCyc

A biofilm was created by culturing Pseudomonas aeruginosa (ATCC 27853)for 24 hours in a well at 37° C. grZ14s-nvCyc peptide at differentconcentrations was subsequently added to the well and allowed toincubate for 24 hours. The Abac10-cyc peptide served as a negativecontrol. The P.a. control was Pseudomonas aeruginosa alone. FIG. 5demonstrates that grZ14s-nvCyc peptides caused far greater microorganismdetachment than in the P.a. control or negative control. Staining wasperformed with crystal violet at OD 595 nm.

Example 7 Detaching 24-Hour Biofilm with Overnight PhyscoZ-Cyc

A biofilm was created by culturing Pseudomonas aeruginosa (ATCC 27853)for 24 hours in a well at 37° C. PhyscoZ-Cyc peptide [Custom PeptideSynthesis; Peptron, Korea] at different concentrations was subsequentlyadded to the well and allowed to incubate for 24 hours. The P.a. controlwas Pseudomonas aeruginosa alone. FIG. 6 demonstrates that PhyscoZ-Cycpeptides caused far greater microorganism detachment than in the P.a.control. Staining was performed with crystal violet at OD 595 nm.

Example 8 grZ14s-nvCyc Enhances Antibiotic Activity of Imipenem andAmpicillin

The ability of grZ14s-nvCyc to enhance the activity of Imipenem orAmpicillin was performed by first incubating the wells in a 96-wellplate with Pseudomonas aeruginosa for 24 hours at 37° C. and 50 rpmshaking. The plate was washed 2 times with phosphate buffer solution.The wells were then filled with either the antibiotic alone or theantibiotic with grZ14s-nvCyc (100 ng/ml). The wells were then incubatedfor 48 hours (24 hours for Ampicillin) at 37° C. and 50 rpm shaking.0.3% triton X-100 and 0.45% EDTA were then added to the wells, bringingthe wells to a final concentration of 0.075% triton X-100 and 0.1125%EDTA. The plates were then sonicated in a bath-sonicator for 12 minutes.The wells were then serially diluted in PBS. The solutions were thenseeded on a blood plate agar and incubated at 37° C. for 24 hours.

The results are presented in FIGS. 7 and 8. As FIG. 7 demonstrates, thenumber of live bacteria was much lower in wells with grZ14s-nvCyc andImipenem than in wells with Imipenem alone, thereby demonstrating thatgrZ14s-nvCyc enhances the activity of the antibiotic. FIG. 8demonstrates that the number of live bacterial is much lower in wellswith grZ14s-nvCyc and Ampicillin than in wells with Ampicillin alone,thereby demonstrating that grZ14s-nvCyc enhances the activity of theantibiotic.

Example 9 grZ14s-nvCyc Enhances Antibiotic Activity of Vancomycin

The ability of grZ14s-nvCyc to enhance the activity of Vancomycin wasperformed by first incubating the wells in a 96-well plate withStaphylococcus aureus for 24 hours at 37° C. and 50 rpm shaking. Theplate was washed 2 times with phosphate buffer solution. The wells werethen filled with either the antibiotic alone or the antibiotic withgrZ14s-nvCyc (100 ng/ml). The wells were then incubated for 24 hours at37° C. and 50 rpm shaking. 0.3% triton X-100 and 0.45% EDTA were thenadded to the wells, bringing the wells to a final concentration of0.075% triton X-100 and 0.1125% EDTA. The plates were then sonicated ina bath-sonicator for 12 minutes. The wells were then serially diluted inPBS. The solutions were then seeded on a blood plate agar and incubatedat 37° C. for 24 hours.

The results are presented in FIG. 9. As the demonstrates, the number oflive bacteria was much lower in wells with grZ14s-nvCyc and Vancomycinthan in wells with Vancomycin alone, thereby demonstrating thatgrZ14s-nvCyc enhances the activity of the antibiotic.

Example 10 grZ14s-nvCyc Enhances Antifungal Activity of Amphotericin

The ability of grZ14s-nvCyc to enhance the activity of Amphotericin wasperformed by first incubating the wells in a 96-well plate with Candidaalbicans for 24 hours at 37° C. and 50 rpm shaking. The plate was washed2 times with phosphate buffer solution. The wells were then filled witheither the antifungal alone or the antifungal with grZ14s-nvCyc (100ng/ml). The wells were then incubated for 24 hours at 37° C. and 50 rpmshaking. The plates were again washed 2 times with PBS. BacTiter-GloMicrobial Cell Viability Assay (Promega, USA) was used following themanufacturer protocol and luminescence read.

The results are presented in FIG. 10. As the demonstrates, the number oflive fungus was much lower in wells with grZ14s-nvCyc and Amphotericinthan in wells with Amphotericin alone, thereby demonstrating thatgrZ14s-nvCyc enhances the activity of Amphotericin.

Example 11 grZ14s-nvCyc Enhances Antifungal Activity of Fluconazole

The ability of grZ14s-nvCyc to enhance the activity of Fluconazole wasperformed by first incubating the wells in a 96-well plate with Candidaalbicans for 24 hours at 37° C. and 50 rpm shaking. The plate was washed2 times with phosphate buffer solution. The wells were then filled witheither the antifungal alone or the antifungal with grZ14s-nvCyc (100ng/ml). The wells were then incubated for 24 hours at 37° C. and 50 rpmshaking. The plates were again washed 2 times with PBS. BacTiter-GloMicrobial Cell Viability Assay (Promega, USA) was used following themanufacturer protocol and luminescence read.

The results are presented in FIG. 11. As the demonstrates, the number oflive fungus was much lower in wells with grZ14s-nvCyc and Fluconazolethan in wells with Fluconazole alone, thereby demonstrating thatgrZ14s-nvCyc enhances the activity of Fluconazole.

Example 12 grZ14s-nvCyc Enhances Antibiotic Activity of Kanamycin

The ability of grZ14s-nvCyc to enhance the activity of Kanamycin wasperformed by first incubating the wells in a 96-well plate withPseudomonas aeruginosa for 24 hours at 37° C. and 50 rpm shaking. Theplate was washed 2 times with phosphate buffer solution. The wells werethen filled with either Kanamycin alone or Kanamycin with grZ14s-nvCyc(100 ng/ml). The wells were then incubated for 24 hours at 37° C. and 50rpm shaking. The plates were again washed 2 times with PBS. BacTiter-GloMicrobial Cell Viability Assay (Promega, USA) was used following themanufacturer protocol and luminescence read.

The results are presented in FIG. 12. As the demonstrates, the number oflive bacteria was much lower in wells with grZ14s-nvCyc and Kanamycinthan in wells with Kanamycin alone, thereby demonstrating thatgrZ14s-nvCyc enhances the activity of Kanamycin.

Example 13 Preventing Adhesion and Detachment of Microorganisms withVarious Peptides

The peptides depicted in Table 1 (each with a cysteine attached to theN- and C-terminus of the peptides) were synthesized using solid-phasemethods and were purified to 90% (Peptron, Inc.; Taejeon, South Korea).The peptides were dissolved in 20 μl dimethyl sulfoxide and diluted indouble distilled water to a concentration of 10 mg/ml. Additionaldilutions were performed in phosphate buffered saline (PBS).

The ability of these peptides to prevent microbial adhesion to a surfaceor detach microbial adhesion from a surface were measured against thefollowing microbial strains: Pseudomonas aeruginosa (ATCC27853),Staphylococcus aureus (ATCC25923), Candida albicans (ATCC14053) andAcinetobacter baumannii (clinical isolate). Specifically, peptidesdiluted to concentrations of 100 ng/ml were incubated with the foregoingmicrobes for 24 hours.

Biofilms of the foregoing microorganisms were grown in a 96-wellfound-bottom polystyrene plate. Specifically, 180 μl of overnightcultures were added to the wells with 20 μl of each peptide diluted inPBS, either simultaneously to measure prevention or after a period oftime to measure detachment from 4 hours up to 24 hours post incubation.After 24 hours of incubation at 37° C., each well was washed with waterand stained with 250 μl of crystal violet solution. The crystal violetsolution was then removed by thoroughly washing the wells with water. Toquantify the number of bacteria cells attached to the wells, the crystalviolet was solubilized in 250 μl of 1% sodium dodecyl sulfate and theabsorbance was measured at 595 nm. As Table 1 demonstrates, the peptidesdecreased the amount of microbial attachment to the wells (Table 1:“Biofilm prevention”) and promoted microbial detachment (Table 1:“Detachment), thereby preventing the formation of biofilm by themicroorganisms and causing them to detach where they have alreadyadhered to a surface.

TABLE 1 Biofilm prevention and detachment by various peptides BiofilmDetachment prevention (%) (%) (100 ng/ml) Peptide: SelagZ-cycCSVPFDYNLYSNWC (SEQ ID NO: 112) Pseudomonas aeruginosa 88% 67% ATCC27853(100 ng/ml) Staphylococcus aureus 88% 35% ATCC25923 (100 ng/ml)Candida albicans 80% NA ATCC14053  (10 ng/ml) Acinetobacterbaumannii 80%50% (100 ng/ml) Peptide: PhyscoZ-cyc CSVPFDYNLYSNWC (SEQ ID NO: 112)Pseudomonas aeruginosa 95% 75% ATCC27853 (100 ng/ml)Staphylococcus aureus 65% 70% ATCC25923 (100 ng/ml) Candida albicans 62%NA ATCC14053  (10 ng/ml) Acinetobacterbaumannii NA 50%Peptide: EchoZ-cyc CSAPYNFNFYSNWC (SEQ ID NO: 113)Pseudomonas aeruginosa 90% 65% ATCC27853 (100 ng/ml)Staphylococcus aureus 85% 35% ATCC25923 (100 ng/ml) Candida albicans 80%NA ATCC14053  (10 ng/ml) Acinetobacterbaumannii 80% 60% (100 ng/ml)Peptide: XenoZ-S-cyc CSRYSSFDYDWYNVC (SEQ ID NO: 114)Pseudomonas aeruginosa 57% 30% ATCC27853  (10 ng/ml)Staphylococcus aureus 65% NA ATCC25923 (100 ng/ml) Candida albicans 75%50% ATCC14053 (100 ng/ml) Acinetobacterbaumannii 95% 49% (100 ng/ml)Peptide:CionaZ-S-cyc CSELSSFNFDWYNVC (SEQ ID NO: 115)Pseudomonas aeruginosa 75% 78% ATCC27853 (100 ng/ml)Staphylococcus aureus 90% NA ATCC25923 (100 ng/ml) Candida albicans 68%42% ATCC14053 (100 ng/ml) Acinetobacterbaumannii 52% 80% (100 ng/ml)Peptide: CanisZ-cyc CNVHSFDYDWYNVC (SEQ ID NO: 116)Pseudomonas aeruginosa 95% 72% ATCC27853 (100 ng/ml)Staphylococcus aureus 75% NA ATCC25923 (100 ng/ml) Candida albicans 80%45% ATCC14053 (100 ng/ml) Acinetobacterbaumannii 60% 73%  (10 ng/ml)Peptide: NilotiZ-cyc CRVESFNYDWYNVC (SEQ ID NO: 117)Pseudomonas aeruginosa 90% 40% ATCC27853 (100 ng/ml)Staphylococcus aureus 63% 65% ATCC25923 (100 ng/ml) Candida albicans 30%65% ATCC14053 (100 ng/ml) Acinetobacterbaumannii 87% NA (100 ng/ml)Peptide: SalmoZ-cyc CRVESFDFDWYNIC (SEQ ID NO: 118)Pseudomonas aeruginosa 90% 80% ATCC27853  (10 ng/ml)Staphylococcus aureus 80% 75% ATCC25923 (100 ng/ml) Candida albicans 55%73% ATCC14053 (100 ng/ml) Acinetobacterbaumannii 95% NA (100 ng/ml)Peptide: TetraoZ-cyc CRINSFDYDWYNVC (SEQ ID NO: 119)Pseudomonas aeruginosa 45% NA ATCC27853 (100 ng/ml)Staphylococcus aureus 52% 43% ATCC25923 (100 ng/ml) Candida albicans 25%55% ATCC14053 (100 ng/ml) Acinetobacterbaumannii 70% NA  (10 ng/ml)Peptide: AnoliZ-cyc CTVNSFDYDWYNVC (SEQ ID NO: 120)Pseudomonas aeruginosa 95% 90% ATCC27853 (100 ng/ml)Staphylococcus aureus 75% 72% ATCC25923 (100 ng/ml) Candida albicans 55%72% ATCC14053 (100 ng/ml) Acinetobacterbaumannii 92% NA  (10 ng/ml)Peptide: MeleagZ-cyc CKVNSFDYDWYNVC (SEQ ID NO: 121)Pseudomonas aeruginosa 82% 65% ATCC27853 (100 ng/ml)Staphylococcus aureus 73% 80% ATCC25923 (100 ng/ml) Candida albicans 30%62% ATCC14053 (100 ng/ml) Acinetobacterbaumannii 95% NA (100 ng/ml)Peptide: CapraZ-cyc CTVHSFDYDWYNVC (SEQ ID NO: 122)Pseudomonas aeruginosa 68% 65% ATCC27853 (100 ng/ml)Staphylococcus aureus 42% 55% ATCC25923 (100 ng/ml) Candida albicans 25%60% ATCC14053 (100 ng/ml) Acinetobacterbaumannii 90% NA (100 ng/ml)

Peptides listed in Table 2, synthesized in the same manner describedabove for Table 1, have a cysteine that was added to the N- andC-terminal ends of the peptides, as represented by the “C” at either endof the peptides. These peptides also are cyclized, as indicated by the“(C-C)” term at the end of each peptide. EqSyn-Neg- and GRSyn-Neg wereadded as negative controls.

TABLE 2 Various peptides Sequence Sequence (SEQ ID NOS 123-141,(SEQ ID NOS 142-156, Peptide respectively, in  Peptide respectively, inNo ID order of appearance) No ID order of appearance)  1 EqSyn 1CNIPFNFSLNKERC (C-C) 20 GRSyn 1 CSVHSWDYDWYNVC (C-C)  2 EqSyn 2CSVPYQYNWYSNWC (C-C) 21 GRSyn 2 CSVHSYDFDWYNVC (C-C)  3 EqSyn 3CSVPWEYNFYSNWC (C-C) 22 GRSyn 3 CTLQAFNYEWYQLC (C-C)  4 EqSyn 4CRIPYDRGMIVNVC (C-C) 23 GRSyn 4 CKYETFEYGWYNIC (C-C)  5 EqSyn 5CKVPYDWDSVINLC (C-C) 24 GRSyn 5 CHGDSFQYEWYNLC (C-C)  6 EqSyn 6CQLPYDVHTYNDWC (C-C) 25 GRSyn 6 CSVHSFDWDWYNVC (C-C)  7 EqSyn 7CLAPYDHNRYTQWC (C-C) 26 GRSyn 7 CSVHSFDYDYYNVC (C-C)  8 EqSyn 8CSNPYDLEAYENWC (C-C) 27 GRSyn 8 CSVHSFDYDFYNVC (C-C)  9 EqSyn 9CSVPYDYQGYRNIC (C-C) 28 GRSyn 9 CSVHSFDYDWFNVC (C-C) 10 EqSyn 10CSVPYDYNVYLNKC (C-C) 29 GRSyn 10 CSVHSFDYDWWNVC (C-C) 11 EqSyn 11CIQPYDKNYFQNFC (C-C) 30 GRSyn 11 CIFNPFDYDWYNVC (C-C) 12 EqSyn 12CVVPYDINIKDNWC (C-C) 31 GRSyn 12 CQWHSFDYDWYNVC (C-C) 13 EqSyn 13CSVPYDYNPYSNWC (C-C) 32 GRSyn 13 CDVHPFDYDWYNVC (C-C) 14 EqSyn 14CSVPYDYNKLKNWC (C-C) 33 GRSyn-Neg CSVHSGDYDGGNVC (C-C) 15 EqSyn 15CSVPYDYNWRSSWC (C-C) 34 grZ14s-nyCyc CSVHSFDYDWYNVC (C-C) 16 EqSyn 16CSVPYDYNWWSAWC (C-C) 17 EqSyn 17 CSVPYDYNWQSNWC (C-C) 18 EqSyn-NegCSVGYDYNWYSNWC (C-C) 19 Eqt2Z-cyc CSVPYDYNWYSNWC (C-C)

The ability of these peptides to (1) prevent microbial adhesion to asurface and to (2) detach microbes already adhered to a surface weremeasured against the following microbial strains: Pseudomonas aeruginosa(ATCC27853), Staphylococcus aureus (ATCC25923), Candida albicans(ATCC14053) and/or Escherichia coli. The resulting percentage ofprevention or detachment, relative to control wells in which themicrobes was allowed to culture in the absence of peptides, areindicated in FIGS. 13 to 16. The figures demonstrate that these peptidesare able to prevent microbial adhesion to a surface and detach microbesalready adhered to a surface.

Example 14 Preventing Adhesion of Pseudomonas aeruginosa with PeptidesModified from Eqt2Z-cyc

The Eqt2Z-cyc peptide was modified by inserting synthetic amino acidanalogs in different positions in the natural sequence. Table 3 depictsthe modifications performed on Eqt2Z-cyc. The modified peptides wereincubated with Pseudomonas aeruginosa. A biofilm was created byculturing the microorganism for 24 hours in a well at 37° C., modifiedEqt2Z-cyc-mod peptide was subsequently added to the well and allowed toincubate for 24 hours. FIG. 17 depicts the results of the incubation, inwhich the peptides prevented adhesion of the microorganism when comparedwith incubation of the microorganism without any peptide.

TABLE 3 Modifications of Eqt2Z-cyc Eqt2ZCyc-mod1(D-form Cys)-SVPYDYNWYSNWC (C-C) Eqt2ZCyc-mod2CSVPYDYNWYSNW-(D-form Cys) (C-C) Eqt2ZCyc-mod3(D-form Cys)-SVPYDYNWYSNW- (D-form Cys) (C-C) Eqt2ZCyc-mod4homoCys-SVPYDYNWYSNWC (C-C) (SEQ ID NO: 157) Eqt2ZCyc-mod5CSVPYDYNWYSNW-homoCys (C-C) (SEQ ID NO: 158) Eqt2ZCyc-mod7CS-(D-form V)-PYDYNWYSNWC (C-C) Eqt2ZCyc-mod8CSV-(D-form P)-YDYNWYSNWC (C-C)

Example 15 Peptides Contribute to Detachment of Microorganisms byAffecting Polysaccharide Matrix Production

In one embodiment, peptides can cause microorganisms to detach from asurface by affecting the microorganisms' production of polysaccharidematrix. The effect of Eqt2Z-Cyc and grZ14s-nvCYC on the polysaccharidematrix production of Pseudomonas aeruginosa, Candida albicans andStaphylococcus aureus were evaluated by measuring Congo Red and/orTrypan Blue after the peptides were incubated with the microorganisms.Congo Red and Trypan Blue are dyes that bind to the polysaccharidematrix via exopolysaccharide fibrils. A reduction in absorbance of thesedyes corresponds with a reduction in polysaccharide matrix production.

Specifically, Congo Red and Trypan blue binding assays were performed bygrowing Pseudomonas aeruginosa, Staphylococcus aureus and Candidaalbicans in specific growth media (LB, TSB+0.25% glucose, RPMI 1640,respectively) simultaneously with the peptides until they reached adensity of OD_(600nm)=0.25. The cells were then pelleted, the supernantremoved and the cells were resuspended in TMP buffer (contains 10.0 mMTris/HCl (pH 8.0), 1 mMKH₂PO₄ and 8.0 MM MgSO₄) to a density ofOD_(600nm)=0.25. Aliquots of the cell suspensions were mixed with stocksolutions of Congo Red (150 μg ml⁻¹) and Trypan Blue (100 ml⁻¹). TPMbuffer was added to the cell/dye mixtures to give final concentrationsof 2.5×10⁸ cells ml⁻¹ and either 15 μg Congo Red ml⁻¹ or 10 μg TrypanBlue ml⁻¹. Cell-free samples containing TPM buffer and 15 μg Congo Redml⁻¹ or 10 μg Trypan Blue ml⁻¹ were used as controls. All samples werevortexed briefly and incubated in a 25° C. dark room for 30 min.Following the incubation, the cells were pelleted, and the supernatantswere transferred to cuvettes. The absorbance of each supernatant samplewas measured at 490 nm to detect Congo Red or at 585 nm to detect TrypanBlue, and these values were compared to the absorbance of theappropriate control sample. Each test sample and control sample wasanalysed three times.

The results are depicted in FIGS. 18-23. As the figures demonstrate,both peptides contributed to a reduced polysaccharide matrix productionin each of the microorganisms, evident by the reduced Congo Red andTrypan Blue absorbance coming from cells incubated with the peptides ascompared to cells that were not incubated with the peptides.

1. A peptide consisting of amino acidsX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 1), wherein X¹ is S,N, I, V, R, K, Q or L; X² is V, I, A, N, L or Q; X³ is P; X⁴ is Y, F orW; X⁵ is D, N, Q or E; X⁶ is Y, F, R, W, V, H, L, K or I; X⁷ is N, S, G,D, H, E, Q or I; X⁸ is W, L, F, M S, T, R, A, G, V, P, Y, I or K; X⁹ isY, N, F, K, L, R, V, W or Q; X¹⁰ is S, K, N, T, E, R, L, Q, I, V, D, orK; X¹¹ is N, E, D, Q, S, A or I; and X¹² is W, R, V, L, I, K, F or E,wherein the peptide is not SVPYDYNWYSNW (SEQ ID NO: 2). 2-57. (canceled)58. The peptide of claim 1, wherein the peptide is SVPFDYNLYSNW (SEQ IDNO: 5); SAPYNFNFYSNW (SEQ ID NO: 6); NIPFNFSLNKER (SEQ ID NO: 7);SVPYQYNWYSNW (SEQ ID NO: 8); SVPWEYNFYSNW (SEQ ID NO: 9); RIPYDRGMIVNV(SEQ ID NO: 10); KVPYDWDSVINL (SEQ ID NO: 11); QLPYDVHTYNDW (SEQ ID NO:12); LAPYDHNRYTQW (SEQ ID NO: 13); SNPYDLEAYENW (SEQ ID NO: 14);SVPYDYQGYRNI (SEQ ID NO: 15); SVPYDYNVYLNK (SEQ ID NO: 16); IQPYDKNYFQNF(SEQ ID NO: 17); VVPYDINIKDNW (SEQ ID NO: 18); SVPYDYNPYSNW (SEQ ID NO:19); SVPYDYNKLKNW (SEQ ID NO: 20); SVPYDYNWRSSW (SEQ ID NO: 21);SVPYDYNWWSAW (SEQ ID NO: 22); SVPYDYNWQSNW (SEQ ID NO: 23).
 59. Thepeptide of claim 1, wherein said peptide is a cyclic peptide.
 60. Acomposition comprising a peptide according to claim
 1. 61. A method ofdetaching a single cell organism from a surface or from other singlecell organisms, comprising contacting said organism with a compositionaccording to claim
 60. 62. A method of dispersing a biofilm or detachingbiofilm formation from a surface, comprising treating water with orcoating said surface with a composition according to claim
 60. 63. Amethod of increasing the effectiveness of a pharmaceutical composition,the method comprising administering to a subject in need thereof acomposition according to claim
 60. 64. A cyclic peptide consisting of:a) an amino acid sequence X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ IDNO: 1), wherein X¹ is S, N, I, V, R, K, Q or L; X² is V, I, A, N, L orQ; X³ is P; X⁴ is Y, F or W; X⁵ is D, N, Q or E; X⁶ is Y, F, R, W, V, H,L, K or I; X⁷ is N, S, G, D, H, E, Q or I; X⁸ is W, L, F, M S, T, R, A,G, V, P, Y, I or K; X⁹ is Y, N, F, K, L, R, I, V, W or Q; X¹⁰ is S, K,N, T, E, R, L, Q, I, V, D, or K; X¹¹ is N, E, D, Q, S, A or I; and X¹²is W, R, V, L, I, K, F or E; and b) a cyclization linker between X¹ andX¹², wherein the peptide is not SVPYDYNWYSNW (SEQ ID NO: 2), and whereinthe cyclic peptide has a total length of up to 50 amino acids.
 65. Acomposition comprising a peptide according to claim
 64. 66. A method ofdetaching a single cell organism from a surface or from other singlecell organisms, comprising contacting said organism with a cyclicpeptide consisting of: a) an amino acid sequenceX¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹² (SEQ ID NO: 1), wherein X¹ is S,N, I, V, R, K, Q or L; X² is V, I, A, N, L or Q; X³ is P; X⁴ is Y, F orW; X⁵ is D, N, Q or E; X⁶ is Y, F, R, W, V, H, L, K or I; X⁷ is N, S, G,D, H, E, Q or I; X⁸ is W, L, F, M S, T, R, A, G, V, P, Y, I or K; X⁹ isY, N, F, K, L, R, I, V, W or Q; X¹⁰ is S, K, N, T, E, R, L, Q, I, V, D,or K; X¹¹ is N, E, D, Q, S, A or I; and X¹² is W, R, V, L, I, K, F or E;and b) a cyclization linker between X¹ and X¹², and wherein the cyclicpeptide has a total length of up to 50 amino acids.
 67. A method ofdispersing a biofilm or detaching biofilm formation from a surface,comprising treating water with or coating said surface with acomposition according to claim
 65. 68. A method of increasing theeffectiveness of a pharmaceutical composition, the method comprisingadministering to a subject in need thereof a composition according toclaim
 65. 69. The cyclic peptide according to claim 64, wherein thecyclization linker comprises two amino acids independently selected fromthe group consisting of natural or non-natural amino acids.
 70. Thecyclic peptide according to claim 69, wherein the cyclization linkercomprises two cysteines.
 71. The cyclic peptide according to claim 70,wherein the cyclization linker consists of two cysteines.